Anti-alpha-v integrin antibody for the treatment of fibrosis and/or fibrotic disorders

ABSTRACT

A method can treat a patient suffering from at least one of fibrosis and a fibrotic disorder. The method includes administering a therapeutically effective amount of an anti-αv integrin antibody DI17E6, or a biologically active variant or modification thereof, to the patient.

FIELD OF THE INVENTION

The invention is directed to the treatment of fibrosis and/or fibroticdiseases by means of antibodies. The invention is furthermore directedto the prophylaxis of fibrosis and/or fibrotic diseases by antibodies.Above all, the invention relates to the administration of ananti-alpha-v integrin (receptor) antibody to patients suffering fromfibrosis and/or fibrotic diseases, including but not limited to systemicsclerosis (SSc). More specifically, the instant invention relates to thetreatment of fibrotic diseases of the skin, lung, heart, liver and/orkidney by means of said antibody, and/or the prophylaxis thereof. Evenmore specifically, the instant invention relates to the administrationof a recombinant, de-immunized monoclonal antibody targetingαv-integrins patients suffering from systemic sclerosis, including, butnot limited to systemic sclerosis of the skin, lung, heart and/orkidney. In particular, the invention relates to the therapy of saidpatients by means of the anti-alpha-v integrin antibody DI17E6(Abituzumab) and structural mutants or modifications thereof. Oneimportant target of said therapy is to slow, halt and/or revert saidfibrosis and/or fibrotic diseases in patients, thus preferably togenerally improve the status of a patient suffering from fibrosis and/orfibrotic disease. One further important target of said therapy is toslow, halt and/or revert systemic sclerosis in patients, thus preferablyto generally improve the status and quality of life of the patientsuffering from said systemic sclerosis. Another preferred aspect of theinvention relates to the prophylaxis against fibrosis and/or fibroticdisorders in subjects, preferably human subjects, which are likely todevelop fibrosis and/or fibrotic disorders, by administering theanti-alpha-v integrin antibody DI17E6 (Abituzumab) and/or structuralmutants or modifications thereof.

BACKGROUND OF THE INVENTION

Fibrosis is preferably defined as the formation of excess fibroustissue, preferably fibrous connective tissue, in an organ or tissue,preferably in a reparative or reactive process. This can preferablyqualified as a reactive, benign, or pathological state. In response toinjury, this is preferably called scarring, and if fibrosis arises froma single cell line, this is preferably called a fibroma.Physiologically, fibrosis typically acts to deposit connective tissue,which can obliterate the architecture and function of the underlyingorgan or tissue. Fibrosis can preferably be used to describe thepathological state of excess deposition of fibrous tissue, as well asthe process of connective tissue deposition in healing. In the contextof the present invention, the term fibrosis is preferably used todescribe the pathological state of excess deposition of fibrous tissue.Fibrosis in the pathological sense is similar to the process ofscarring, in that both involve stimulated cells laying down connectivetissue, including collagen and glycosaminoglycans. Immune cells calledmacrophages, as well as any damaged tissue between surfaces calledinterstitium, typically release TGF-β. There are numerous reasons forthis, including inflammation of the nearby tissue, or a generalizedinflammatory state, with increased circulating mediators. TGF-βstimulates the proliferation and activation of fibroblasts, which thennormally trigger the deposition of connective tissue.

Fibrosis can occur in many tissues of many organs within the body,typically as a result of inflammation or damage, and examples include:

Fibrosis of the lung, e.g. pulmonary fibrosis, cystic fibrosis and/oridiopathic pulmonary fibrosis; Fibrosis of the liver, e.g. livercirrhosis;

Fibrosis of the heart, e.g. atrial fibrosis, endomyocardial fibrosisand/or as the consequential damage of a previous myocardial infarction.

Moreover, fibrosis, and especially pathological fibrosis, preferablyincludes arthrofibrosis (predominantly of the knee and shoulder, butalso occurring in a variety of other joints), Crohn's Disease(intestines), Dupuytren's contracture (predominantly in the hands and/orfingers), Keloid (predominantly affecting the skin), Mediastinalfibrosis (predominantly relating to the soft tissue of the mediastinum),Myelofibrosis (predominantly affecting the bone marrow), Peyronie'sdisease (penis), Nephrogenic systemic fibrosis (predominantly affectingthe skin), progressive massive fibrosis (e.g. of the lungs, often aconsequential complication of coal workers' pneumoconiosis),retroperitoneal fibrosis (predominantly affecting the soft tissue of theretroperitoneum), and/or scleroderma or systemic sclerosis(predominantly affecting the skin and/or lungs).

The terms fibrosis, pathological fibrosis and fibrotic diseases orfibrotic disorders are known and understood in the art.

Preferably, all pathological forms of fibrosis, i.e. forms that are notdirectly related to acute damage and/or normal wound healing, are alsoreferred to in the context of the present invention as fibroticdisorders. Thus, fibrotic disorders are preferably those states offibrosis which exceed the level of fibrosis that is normally found indesired, correct wound healing processes.

Systemic sclerosis (SSc, ICD-10 classification M34) is an especiallypreferred fibrotic disorder to be treated according to the instantinvention. Systemic sclerosis is often also referred to as systemicscleroderma and sometimes as progressive systemic sclerosis. Systemicsclerosis is a clinically heterogeneous multi-organ connective tissuedisease with a characteristic but variable spectrum of clinical andlaboratory presentations with features of autoimmunity, vascular injuryand progressive fibrosis, leading to pain, disability, progressivedysfunction and ultimately failure of vital organs such as lung, heart,or kidney. Preferably, SSc can be differentiated from a group ofdiseases termed localized scleroderma, that preferably includeconditions such as morphea, linear scleroderma and sclerodermaen-coup-de-sabre, and preferably also other disorders that mimic one ormore signs of scleroderma.

The aetiology of SSc is currently unknown. However, the spectrum ofclinical presentations is a consequence of variable degrees of vascularabnormalities, immune mediated damage and fibrosis potentially possiblein almost any organ. Organ involvement in SSc can lead to decline of itsfunction and precocious mortality when vital organs such as lung, liver,kidney and heart are affected. The skin is almost always involved. Basedon the pattern of skin involvement SSc is classified into diffusecutaneous (dc) SSc and localized cutaneous (Ic) SSc. In IcSSc skininvolvement typically extends from the distal extremities to the kneesand elbows; in dcSSc the skin involvement typically extends proximally,involving the trunk upper arms and/or thighs. More details on preferredsubsets and disease classifications can be found in the sections“Classification” and “Diagnosis and Symptoms”. SSc can have overlappingfeatures with other connective tissue diseases (CTD) such as systemiclupus erythematosus, polymyositis, rheumatoid arthritis or Sjögren'ssyndrome.

SSc is typically associated with one or more of the followinghistopathological and pathophysiological characteristics:

i) Vascular Abnormalities:

The characteristic pathologic finding in SSc vascular abnormalities is anon-inflammatory proliferative/obliterative vasculopathy involving smallarteries and arterioles in multiple vascular beds. Although inlong-standing SSc these lesions generally occur in the absence ofinflammation, in early stage disease, inflammatory cell infiltrates areprominent in many organs. Histopathologic evidence of vascular damage ispresent before fibrosis can be detected in involved and non-involvedskin, indicating a generalized process. Manifestations, such asRaynaud's phenomenon, generally precede other disease manifestations.Additional clinical signs of SSc vasculopathy include cutaneoustelangiectasia, nail-fold capillary alterations, pulmonary arterialhypertension (PAH), digital pit formation, gastric antral vascularectasia, and scleroderma renal crisis.

In patients with established SSc, the most characteristic vascularfinding is bland intimal proliferation in the small and medium sizedarteries. In late stages of the disease, extensive fibrin deposition andperivascular fibrosis cause progressive luminal occlusion, and there isa striking paucity of small blood vessels in lesional tissue. Loss ofvascular supply leads to chronic tissue hypoxia.

The initial vascular insult is apparently endothelial cell injury.Secondarily platelets may become activated and release mediators thatmay contribute to vasoconstriction, fibroblast activation andmyofibroblast transdifferentiation. Endothelial dysfunction may lead toabnormal vascular dilation/constriction resulting in impaired blood flowresponses and episodes of ischemia-reperfusion with oxidative stressthat amplifies vascular injury. Endothelin-1, the most potentvasoconstrictor known, is reported to be elevated in patients with SSc,with higher levels in dcSSc than IcSSc. Obstructive vasculopathy ofsmall blood vessels leads to tissue hypoxia and the described tissueremodelling. Vasculogenesis may be impaired in SSc and contribute to theprogressive loss of blood vessels.

ii) Tissue Fibrosis:

Fibrosis is characterized by accumulation of excessive amounts of type Icollagen and other fibrillar collagens, fibronectin, elastin,proteoglycans, and other connective tissue molecules in theextracellular matrix (ECM). The process causes disruption of tissuearchitecture. In SSc, interstitial and vascular fibrosis in the skin andinternal organs contributes directly to their progressive dysfunctionand eventual failure. Most prominently affected are the lungs,gastro-intestinal tract, heart, tendon sheath, and perifascicular tissuesurrounding skeletal muscle.

Fibrosis in the skin, the hallmark of SSc, causes marked expansion ofthe dermis. The process obliterates the hair follicles, sweat glands,and other skin appendages. Collagen fiber accumulation is most prominentin the deep dermis, and gradually invades the subadjacent adipose layerwith entrapment of fat cells. The proportion of α-smooth muscleactin-positive myofibroblasts that are intermediates between fibroblastsand contractile smooth muscle cells and play a major role infibrogenesis, is increased in the lesional skin.

In early lung lesions, patchy infiltration of the alveolar walls withlymphocytes, plasma cells, macrophages, and eosinophils is seen. Withprogression, interstitial lung fibrosis and vascular damage predominate,often coexisting within the same lesions. Intimal thickening of thepulmonary arteries underlies PAH, and at autopsy is often associatedwith multiple pulmonary emboli and myocardial fibrosis. The typicalhistologic pattern seen on lung biopsy specimen is non-specificinterstitial pneumonitis, a form of interstitial lung diseasecharacterized by mild-to-moderate interstitial inflammation, type IIpneumocyte hyperplasia, and uniform distribution of fibrosis. Lesscommonly, SSc is associated with the usual interstitial pneumoniapattern, which is characterized by scattered fibroblastic foci andpatchy distribution of fibrosis. Progressive thickening of the alveolarsepta ultimately results in obliteration of the airspaces andhoneycoombing, and consequent loss of pulmonary blood vessels. Thisprocess impairs gas exchange and contributes to increasing pulmonaryarterial tension. The prevalence of interstitial lung disease inpatients with dcSSc is reported to be about 53% and about 35% inpatients with IcSSc.

In the gastrointestinal tract, pathologic changes can occur at any levelfrom the mouth to the rectum. The esophagus is virtually alwaysaffected, with fibrosis in the lamina propria, submucosa, and muscularlayers, and characteristic vascular lesions. Replacement of the normalintestinal architecture results in disordered peristaltic activity,gastroesophageal reflux and small bowel dysmotility, pseudo-obstruction,and bacterial overgrowth. Chronic gastroesophageal reflux is complicatedby esophageal inflammation, ulcerations, and stricture formation.

In the kidneys, vascular lesions predominate, and glomerulonephritis israre. Chronic renal ischemia is associated with shrunken glomeruli andother ischemic changes. Patients with acute scleroderma renal crisisshow dramatic histological changes indistinguishable from other forms ofmalignant hypertension. Vascular changes in SSc kidneys are mostprominent in the small interlobular and arcuate arteries, which showreduplication of elastic lamina, marked intimal proliferation, andaccumulation of ground substance. These changes can also be found in SScpatients who do not have renal crisis. Fibrinoid necrosis of thearteriolar walls may be seen. Intimal thickening leads to severenarrowing and total obliteration of the lumen, often withmicroangiopathic hemolysis.

At autopsy evidence of cardiac involvement is found in 70% of patientswith SSc. Modest pericardial effusions are common; occasionally fibrosiswith constrictive pericarditis may occur. A characteristic pathologicfinding is myocardial contraction band necrosis, which is thought toreflect ischemia-reperfusion injury. Significant interstitial andperivascular fibrosis may occur in the absence of clinically evidentheart involvement.

Other organs with fibrotic alterations include the thyroid, penile bloodvessels associated with erectile dysfunction, salivary and lacrimalglands. Synovial biopsy specimens show fibrosis and characteristicvascular changes in the small arterioles.

The cellular source of excessive deposition of collagen and other ECMconstituents are fibroblast or fibroblast-like cells. Fibroblastsnormally residing in the connective tissue or pericytes residing aroundblood vessels may become activated by growth factors such as TGF-βresulting in proliferation and increased collagen synthesis. Tissueinjury, mechanical tension and TGF-β induce activation offibroblast-like cells and a phenotypic change, resulting in thetransformation of these cells into myofibroblasts, a process designatedfibroblast-myofibroblast-transformation (FMT). Myofibroblasts arecharacterized by increased motility, expression of a smooth muscleactin, increased collagen synthesis, tissue inhibitors ofmetalloproteinases, and other ECM components. Myofibroblasts are a majorsource of TGF-β activation during the fibrotic response and areresponsible for contraction of early granulation tissue. In pathologicfibrogenesis, myofibroblasts persist, resulting in excessivelycontracted ECM characteristic of chronic scars.

Immune Dysfunction:

The innate and adaptive arms of the immune system seem to be activatedin early SSc, and autoimmunity is prominent; however, the role ofcellular and humoral autoimmune effector pathways in the pathogenesis isuncertain.

In early stages of the disease, activated CD4 and CD8 lymphocytes andmonocytes and macrophages, and less commonly B cells, eosinophils, mastcells, and natural killer cells, are observed in perivascular regions inthe lesional skin, lungs, and other affected organs. Mononuclear cellinfiltrates in skin are predominantly CD3CD4 positive T cells andexpress markers of activation.

Circulating autoantibodies with multiple antigenic specificities can bedetected in virtually all patients with SSc.

Although SSc-associated autoantibodies have validated clinical utilityas diagnostic markers, their contribution to disease manifestations isuncertain and it is unknown whether these autoantibodies precede, or area consequence of, vascular injury, tissue damage and/or fibrosis. Targetspecificities and clinical associations are summarized in table 1.

TABLE 1 Autoantibody frequency and their main clinical associationsFrequency Autoantibody type (%) Clinical Associations Antinuclearantibody 93-9  lcSSc and dcSSc Anti-centromere 16-39 lcSSc, PAH withoutILD, PBC, protective for ILD abnd SRV Anti-topoisomerase 1  9-39 dcSSc >lcSSc, ILD, SDV Anti-RNA polymerase  4-25 dcSSC, SRC Anti-Th/to 1-7lcSSc, ILD, PAH Anti-U3RNP 1-6 dcSSc > lcSSc, severe disease, muscleinvolvement, PAH Anti-PM-Scl 0-6 PM/DM overlap, arthritis overlap, ILDAnti-Ku 1-3 Muscle and joint involvement Anti-U1RNP  5-35 Overlapsyndromes Anti-U11/U12RNP 1.6-5   ILD Abbreviations: PBC, primarybiliary cirrhosis; PM/DM, polymyositis/dermatomyositis; SDV, severedigital vasculopathy; SRC, scleroderma renal crisis.

SSc is typically associated with one or more of the following clinicalcharacteristics: The clinical manifestations of SSc are protean,reflecting its complex underlying pathology. The frequency of variousclinical features differs according to the stage and subset of thedisease. The course and the severity of organ involvement areunpredictable in individual patients. In addition, the severity andactivity of each complication needs to be considered in making treatmentdecisions. Fatigue and lethargy are common throughout the illness,although usually more pronounced in its early phases. Reactivedepression is a frequent accompaniment to this often relentless anddisfiguring disorder.

The prevalence of major organ manifestations reported in textbooks isgiven in table 2. Higher frequencies were recently published from theEuropean Scleroderma Trials and Research (EUSTAR) cohort based on thecharacteristics of 7,655 patients, all fulfilling the 1980 ACR criteriafor the clinical characteristics of the different involved organ systemsare presented in a tabulated overview (tables 2 and 3). Typically,females are affected more often than males, with a predominance of 3-5:1being reported.

TABLE 2 Clinical characteristics of SSc Skin In some cases edemainitially (puffy hands and fingers, sometimes feet) followed bythickening and hardening of the skin, sometimes visible skininflammation, sometimes hypo and hyperpigmentation. Distribution of skinlesions: distal extremities in localized cutaneous SSc (lcSSc) or indiffuse cutaneous SSc (dcSSc) with lesions extending proximally andinvolving truncal skin (see also table). Raynaud's phenomenon. Episodicvasospasm induced by cold or emotional stress. Intermittent pallorfollowed by cyanosis, suffusion, or pain and tingling, and sometimesredness. Telangectasias are dilated small blood vessels in the skinforming red spots. Telangiectasias in SSc are typically oval orrectangular in shape. Abnormal nailfold capillaries. Complications: lossof skin appendices (sweat glands, hair follicles) and subcutaneous fat,neural compression (e.g. carpal tunnel syndrome), adherence to tendonsand joints, limited to absent joint motility, digital pitting scars andulcers, digital gangrene, other skin ulcers (e.g. overlying themetacarpophalangeal joints) due to vascular involvement, impaired woundhealing, infection of skin ulcers, calcinosis cutis (macroscopic tissuecalcifications that can break through skin and also lead to chronic skinulceration). Gastrointestinal tract Mouth: perioral tight skin, reducedoral aperture, dental caries, xerostomia. Esophagus: dysmotility,reflux; complications: strictures, hiatal hernia, Barrett's metaplasia(replacement of physiologic squamous epithelium by columnar epitheliumwith goblet cells, precancerous condition). Stomach: gastroparesis withbloating and vomiting, gastric antral vascular ectasia with intermittentbleeding (can cause anemia). Small bowel: hypomotiliy, stasis, bacterialovergrowth; complications: pseudoobstruction, diarrhea, bloating,malabsorption, weight loss, malnutrition, cachexia. Large bowel:hypomotility, pseudodiverticulosis; complications:pseudoobstruction/megacolon, volvulus, pneumatosis cystoidesintestinalis. Rectum: sphincter incompetence. Musculoskeletal systemInflammatory synovitis and tendon friction rubs caused by inflammationin tendon sheath. Fibrotic process in tendons, ligaments and jointcapsules can contribute together with skin fibrosis to jointcontractures. Myopathy, myositis in case of SSc-/myositis overlap.Osteolysis.

TABLE 3 Clinical characteristics of SSc cont. Lung Pulmonary fibrosisand/or alveolitis (interstitial lung disease, ILD) with breathlessness,especially on exertion, dry cough, bilateral inspiratory crackles at thelung bases, radiographic features of ILD (in conventional radiographs,in high resolution computer tomography (HRCT) with higher sensitivityand at earlier stages) and abnormal lung function tests (reduced ForcedVital Capacity, FVC; Total Lung Capacity, TLC; Diffusion Capacity ofLung for carbon monoxide, DLCO). The survival of patients with fullblown SSC-ILD is much shorter than the survival of overall SSC patients.Pulmonary hypertension (1) as a primary abnormality due tofibroproliferative abnormalities in the pulmonary vasculature (pulmonaryarterial hypertension, PAH), (2) associated with disease of the leftside of the heart, (3) associated with chronic hypoxia (due to ILD andloss of pulmonary vascular bed), (4) associated with chronicthromboembolism, including pulmonary occlusive disease. PAH usually doesnot manifest with dyspnea until quiet advanced stages. Reduced exercisecapacity is a typical finding. PAH can be characterized by abnormalechocardiographic, pulmonary function and/or electrocardiographicfindings, although right heart catheterization remains the gold standardand is required to confirm the diagnosis of PAH. Definitive diagnosisrequires exclusion of thromboembolic disease, >25 mm Hg of the meanpulmonary arterial pressure at rest or >30 mm Hg with exercise. Theprognosis of PAH associated with SSc is worse than idiopathic pulmonaryarterial hypertension. Aspiration pneumonitis. Pleural effusions,pleuritis. Bronchiectasis. Lung cancer. Heart Myocardial enzymeelevation, ECG abnormalities including abnormalities of cardiac rate andrhythm, diastolic or global dysfunction as a consequence of myocardialischemia, fibrosis, and/or myocarditis. Pericardial effusion.Complications: left ventricular or global heart failure, myocardialinfarction, sudden death. Kidney Indolent chronic renal involvement withslow reduction of glomerular filtration rate and proteinuria.Glomerulonephritis in case of SSc-systemic lupus erythematosus overlap.Complication: scleroderma renal crisis characterized by severe arterialhypertension, that can cause heart failure, stroke or encephalopathywith generalized seizures, flash pulmonary edema, and progressive oracute renal failure with increased creatinine serum levels, proteinuria,microscopic hematuria, and sometimes microangiopathic haemolytic anemiaand thrombocytopenia, can evolve into end stage renal disease requiringlong-term dialysis or renal transplantation, early mortality inapproximately 10%.

Classification:

The hallmark of SSc is induration and thickening of the skin(“scleroderma”), but also many internal organs can be involved in SSc.The two major clinical subsets are differentiated by the pattern ofcutaneous involvement and additional associated clinical and laboratoryfeatures (Table 4). The CREST syndrome (acronym derived from calcinosiscutis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly,telangiectasias) has been individualized based on a combination ofclinical features but may be classified as IcSSC. SSc can have featuresof other connective tissue diseases or fulfil their criteria. SScwithout skin involvement (“scleroderma sine scleroderma”) is rare andusually diagnosed late in the course due to absent skin signs. SSc inchildhood and adolescence is extremely rare. Further classifications canbe found in the section titled “Diagnosis and Symptoms”.

TABLE 4 Key clinical features of SSc subsets Diffuse cutaneous systemicsclerosis (dcSSc) Proximal skin thickening involving the trunk, upperarms and thighs, in addition to symmetrical involvement of the fingers,hands, arms, face/neck. Rapid onset of disease following the appearanceof Raynaud's phenomenon. Significant visceral disease: lungs, heart,gastrointestinal, and/or kidneys. Absence of anti-centromere antibodies.Variable disease course but overall poor prognosis, with survival of 40%to 60% at 10 years. Limited cutaneous systemic sclerosis (lcSSc)Symmetrical skin thickening limited to the areas below the elbows andknees and involving the face/neck. Progression if disease typicallymonths or years after the onset of Raynaud's phenomenon. Later and lesssevere development of visceral disease. Late development of pulmonaryarterial hypertension. Association with anti-centromere antibodies.Relatively good prognosis with survival >70% at 10 years. Overlapsyndromes Diffuse or limited systemic sclerosis with typical features ofone or more of the other defined connective tissue diseases. Mixedconnective tissue disease: features of systemic lupus erythematosus,systemic sclerosis and polymyositis in the presence of anti-U1 RNPantibodies. CREST syndrome Subset of lcSSc with prominent calcinosis,Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, andteleangiectasis (CREST). Scleroderma sine scleroderma (SSc without skininvolvement) Raynaud's phenomenon, characteristic internal organcomplications, and serologic abnormalities of SSc, but no apparent skinthickening and stiffening.

Diagnosis and Symptoms:

The diagnosis of SSc is usually made based on clinical manifestations,in particular the pattern of skin involvement. The American College ofRheumatology (ACR) has proposed diagnostic criteria to classifypatients. Either one major criterion (i.e. proximal scleroderma) or twoor more of the minor criteria (i.e. [1] sclerodactyly, [2] digitalpitting scars of fingertips or loss of substance of the distal fingerpad, [3] bilateral basilar pulmonary fibrosis) are required to classifypatients as SSc.

When applied to case and disease comparison cohorts, the criteria had97% sensitivity for definitive SSc and 98% specificity. These criteriaappear not to include all patients with SSc. In the EUSTAR (EULARScleroderma Trials and Research group) cohort only 83.5% of patientsfulfilled the 1980 ACR criteria, patients with early disease and overlapsyndromes comprising the largest proportion of excluded group.

To overcome this issue, the ACR and the European League AgainstRheumatism (EULAR) have agreed on revised criteria for SSc that shouldbe published in the course of 2013. The revised criteria are given inFIG. 1.

A tissue biopsy is preferably not required for the diagnosis of SSc.

TABLE 5 The ACR-EULAR Criteria for the classification of SystemicSclerosis 1. These criteria are applicable to any patient considered forinclusion in a SSc study. 2. These criteria are not applicable topatients having a systemic sclerosis-like disorder better explainingtheir manifestations, such as: nephrogenic sclerosing fibrosis,scleredema diabeticorum, scleromyxedema, erythromyalgia, porphyria,lichen sclerosis, graft versus host disease, and diabeticchierarthropathy. Patients with “Skin thickening sparing the fingers”also are not classified as having SSc. Weight/ Items Sub-items ScoreSkin thickening of the fingers of both hands 9 extending proximal to themetacarpophalangeal joints Skin thickening of the fingers Puffy fingers2 (only count the highest score) Whole Finger, distal to MCP 4 Fingertip lesions Digital Tip Ulcers 2 (only count the highest score) PittingScars 3 Telangiectasia 2 Abnormal nailfold capillaries 2 Pulmonaryarterial hypertension and/or 2 Interstitial lung Disease Raynauds'sphenomenon 3 Scleroderma related antibodies 3 (any of anti-centromere,anti-topoisomerasel [anti-ScL 70], anti-RNA polymerase III) TOTALSCORE{circumflex over ( )}: Patients having a total score of 9 or moreare being classified as having definite systemic sclerosis. {circumflexover ( )}Add the maximum weight (score) in each category to calculatethe total score.

Typical symptoms of SSc are given below in tabulated form (Table 6).

TABLE 6 Symptoms of SSc (clinical signs and other clinical features seeTables 2 and 3) General Fatigue, lethargy Skin Skin sclerosis: sensationof swollen hands or fingers, sometimes pain, sometimes pruritus,impaired manual dexterity/disability in daily private and professionallife due to joint contractures and pain, impaired movement in other thanfinger and hand joints, decreased skin sensitivity due nervecompression, dry skin from loss of skin appendices. Raynaud'sphenomenon: usually painful, sometimes followed by tingling sensation.Skin ulcers and digital gangrene: prolonged episodes of pain, sometimessevere pain. Gastrointestinal tract Mouth: disfigurement. Esophagus:retrosternal discomfort or pain, dysphagia, burning pain/heartburn,regurgitation of gastric material in particular at night, bleeding fromintestinal telangiectasias. Stomach, small/large bowel, and rectum:early satiety, bloating, pain, symptoms of intestinal obstruction,constipation, fecal soiling. Musculoskeletal system Pain due tosynovitis and tendosynovitis. Lung Breathlessness/dyspnea, disabilityfrom breathlessness upon exertion or at rest, cough (typically dry),pain from pleuritis, multiple symptoms due to oxygen therapy and lungtransplantation. Heart Symptoms of myocardial ischemia includingretrosternal pain, palpitations from abnormalities of heart rhythm,symptoms of heart failure. Kidney Headache and blurred vision fromsevere hypertension, neurological symptoms from stroke and seizures,dyspnea from pulmonary edema, multiple symptoms from renal replacement(dialysis, transplantation). Other Symptoms of depression fromrelentless disease, disfigurement and disability, low self-esteem,concerns with physical appearance and feelings about uncertainty aboutthe future.

The average survival time from diagnosis of all SSc patients is reportedto be approximately 13 years, whereas the 5-year survival rate ofpatients with SSc-ILD is reported to be 40-60%, showing the highermortality rate in patients with SSc-ILD compared to overall SSc.

Death in SSc is typically due to SSc-organ involvement (˜53%), cancer(˜15%) or atherosclerosis. Death from SSc-organ involvement is morecommon in patients with diffuse skin involvement, older age at onset,and males.

Recently, the causes and risk factors for death in SSc were reported bythe European League against Rheumatism (EULAR) Scleroderma Trials andResearch (EUSTAR) database). The database included 5,860 SSc patientswho fulfilled the ACR 1980 classification criteria. Causes of death andcomorbidity data were available from 234 of 284 fatalities. Theyreported that 55% of deaths were directly related to SSc and 41% tonon-SSc causes with the remaining 4% of cases considerednonclassifiable. Among the 284 deceased patients, 54.6% had diffusecutaneous disease (dcSSc) and 40.5% had limited cutaneous disease(IcSSc). The median disease duration was 7.1 years for dcSSc and 15years for IcSSc. 19% died of pulmonary fibrosis and 14% of pulmonaryarterial hypertension. SSc-related myocardial disease death was 14% withmost causes being related to arrhythmias. Renal causes of death onlyaccounted for 4%, all of which were related to scleroderma renal crisis.Three percent of patients died from gastrointestinal-related causes.With respect to the non-SSc-related deaths, causes were as follows:infections (13% of all deaths), neoplasia (13%), and cardiovasculardisease (12%). Patients with non-SSc-related deaths were then analyzedfor SSc-related comorbidities. A significant number of patients who diedfrom pneumonia also had presence of gastroesophageal reflux with orwithout documented aspiration. Of the fourteen patients who died fromlung cancer, nine had concomitant pulmonary fibrosis. In this study,independent predictors of reduced survival included presence ofproteinuria, pulmonary arterial hypertension, pulmonary restriction witha forced vital capacity of less than 80% predicted, presence of dyspneagreater than New York Heart Association Class II, higher age at onset ofRaynaud's phenomenon, lower diffusion capacity for carbon monoxide, anda modified Rodman skin score greater than 10. As 35% of all SSc relateddeaths were directly attributable to ILD, and 26% to PAH, this reportreinforced the previous finding that ILD and PAH are the leading causesof SSc-related deaths and likely contribute to non-SSc-related deaths.In a recent systematic review and meta-analysis of 18 studies comprising12,829 patients, the risk of death with cardiac, ILD, pulmonaryhypertension and renal manifestations was elevated).

Presence of ILD is significantly associated with mortality in SSc.Decreased FVC is associated with mortality (VIRGINIA D. STEEN and THOMASA. MEDSGER, JR., ARTHRITIS & RHEUMATISM, Vol. 43, No. 11, November 2000,pp 2437-2444, Assassi et al., Arthritis Rheum. 2009 Oct. 15; 61(10):1403-1411). <70% FVC predicted higher mortality than >70% in SSc-ILD(Goh et al., ARTHRITIS & RHEUMATISM, Vol. 56, No. 6, June 2007, pp2005-2012). Decline in FVC in the preceding 12, 18 and 24 months isbelieved to predict mortality.

In a retrospective study of 953 patients with SSc, patients with severeILD had a 9-year survival rate of approximately 30%, whereas patientswith SSc who did not have severe involvement of an organ system had a9-year survival rate of 72%

Disability is a likewise threatening problem with fibrotic diseases andespecially with SSc. For example, dyspnea is common in SSc (up to 50%).Principal contributing factors include ILD and PAH but bronchiectasis,alveolar hemorrhage, gastroesphageal reflux with aspiration due toesophageal dysmotility, arthritis, obesity, anemia and deconditioningdue to physical inactivity may also contribute.

Dyspnea is a very important and independent predictor of function andhealth-related quality of life (HRQoL). FVC and pulmonary arterysystolic pressure were significant independent predictors of dyspnea.

The disability index (DI) of the modified Health AssessmentQuestionnaire (HAQ) correlates with scleroderma heart, kidney disease,tendon friction rubs, hand contractures, and proximal muscle strength.An increased HAQ-DI is predictive of mortality and correlates withreduced fist closure, reduced hand spread, and presence of tenderjoints. Disability in SSc worsens over time, with dyspnea and diseasetype being the strongest predictor of disability. Patients with digitalulcers have significantly higher global disability, hand disability, andanxiety. Most patients with SSc have limitation in daily activities andhave an increased need for help at home. Skin involvement assessed bythe modified Rodnan Skin Score (mRSS) is strongly associated withdisability and pain. Comparing patients with SSc, psoriatic arthritisand rheumatoid arthritis joint involvement was more disabling in SScthan psoriatic arthritis and SSc patients experienced more pain thanpatients with rheumatoid arthritis. SSc is associated with a highprevalence of depression and anxiety. Depression is associated with ILD.Health related quality of life is reduced in SSc patients and similar torheumatoid arthritis patients. Raynaud's phenomenon has impact ondisability (overall, grip, eating dressing), pain, and mood. Pain anddepressive symptoms are significant determinants of physical functioningand social adjustment.

Working disability and productivity loss is substantial in patients withSSc as concluded in a recent meta-analysis of work status. Standardizedemployment ratios reported were between 0.70 and 0.77 and the proportionof patients being employed ranged between 11.3% and 82%. Full andpart-time sick leave rates are also increased as well as estimated lostproductivity of paid labour. Work disability in SSc is reported togreater than in rheumatoid arthritis.

Thus, there is a very high unmet medical need for treatment options inthe field of fibrosis, fibrotic diseases and especially so in SSc andrelated indications. Furthermore, there is a very high unmet medicalneed for prophylaxis options in order to prevent fibrosis, fibroticdiseases and especially so in SSc and related indications in subjects,preferably human subjects that are likely to develop it.

SUMMARY OF THE INVENTION

It has been found by the inventors that the known monoclonal anti-alphav antibody DI17E6 (designated also as DI-17E, DI17e6, Abituzumab,abituzumab, EMR62242 or EMD 525797) is highly effective in interferingwith cell signalling processes relevant for the development, occurrenceand/or manifestation of fibrosis and especially of fibrotic disorders.Moreover, it has been found by the inventors that said anti-alpha vantibody DI17E6 is highly effective in interfering with cell signallingprocesses relevant for the development, occurrence and/or manifestationof systemic sclerosis. Evidence therefore is shown in the ExperimentalSection given herein and as discussed above and below. Thus, a subjectof the instant invention is the monoclonal anti-alpha αv antibody DI17E6and/or a biologically active variant or modification thereof, for use inthe treatment of fibrosis and/or fibrotic disorders. A preferred subjectof the instant invention is the monoclonal anti-alpha αv antibody DI17E6and/or a biologically active variant or modification thereof, for use inthe treatment of systemic sclerosis. A further subject of the instantinvention is thus the use of the monoclonal anti-alpha αv. antibodyDI17E6 and/or a biologically active variant or modification thereof, forthe manufacture of a medicament for the treatment of fibrosis and/orfibrotic disorders, and especially for the treatment of systemicsclerosis, and/or a method for the treatment of fibrosis and/or fibroticdisorders, and especially for the treatment of systemic sclerosis,comprising administering to a patient the monoclonal anti-alpha αvantibody DI17E6 and/or a biologically active variant or modificationthereof. Due to its favorable safety profile, the monoclonal anti-alphaαv antibody DI17E6 and/or a biologically active variant or modificationthereof are deemed suitable also for the prophylaxis of said disorders.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: Abituzumab Blocks Abituzumab Blocks Elevated aSMA Expression inH358-Fibroblast and Calu3-Fibroblast Co-cultures

FIG. 2 Abituzumab Blocks Elevated Expression of FMT-Related Genes inH358-Fibroblast Co-cultures

FIG. 3 TGF-β Increases Integrins Expression in Human Lung Fibroblast

FIG. 4 TGF-β Increases aSMA, IL-6 and other Myofibroblast Marker GeneExpression in Lung Fibroblast

FIG. 5 Abituzumab Treatment of Fibroblast Cultures Reduces the TGF-βinduced Increase in aSMA and IL-6

FIG. 6 Abituzumab Treatment Reduces TGFb-induced Collagen GelContraction

FIG. 7 Inhibition of αvβ6 6 binding to LAP by Abituzumab in comparisonto anti-HEL-AB MSB0011523H-1 and 10D5, respectively

FIG. 8 Strategy chart for finding the fibrosis/SSc signature

FIG. 9 RGS5 expression by SSc and normal skin in GSE45485 withrespective results of the moderated t-test comparing expression of RGS5in SSc and normal skin

FIG. 10 COL15A1 expression by early IPF, advanced IPF and normal lung inGSE24206 with respective results of the moderated t-test comparingexpression of COL15A1 in early IPF and healthy lung and advanced IPF andhealthy lung, respectively

FIG. 11 COL1A1 expression by early IPF, advanced IPF and normal lung inGSE24206 with respective results of the moderated t-test comparingexpression of COL1A1 in early IPF and healthy lung and advanced IPF andhealthy lung, respectively

FIG. 12 COMP expression by IPAH (PPH), IPF, SSc-PAH, SSc-PF and normallung (NL) in GSE48149 with respective results of the moderated t-testcomparing expression of COMP in early IPF and normal lung and SSc-PF andnormal lung, respectively

FIG. 13 IGFBP2 expression by IPAH (PPH), IPF, SSc-PAH, SSc-PF and normallung (NL) in GSE48149 with respective results of the moderated t-testcomparing expression of IGFBP2 in early IPF and normal lung and SSc-PFand normal lung, respectively

FIG. 14a SSP1 expression by IPAH (PPH), IPF, SSc-PAH, SSc-PF and normallung (NL) in GSE48149 with respective results of the moderated t-testcomparing expression of SSP1 in early IPF and normal lung and SSc-PF andnormal lung, respectively

FIG. 14b Signature Score of 19-gene fibrosis/SSc signature by SSc andnormal skin in GSE45485 with results of the one-sided t-test comparing19-gene Signature Score in SSc and normal skin

FIG. 15a Signature Score of 19-gene fibrosis/SSc signature by SSc andnormal skin in GSE32413 with results of the one-sided t-test comparing19-gene Signature Score in SSc and normal skin

FIG. 15b Signature Score of 9-gene TUAD signature by SSc and normal skinin GSE32413 with results of the one-sided t-test comparing 9-geneSignature Score in SSc and normal skin

FIG. 16a Signature Score of 19-gene fibrosis/SSc signature by SSc andnormal skin in GSE9285 with results of the one-sided t-test comparing19-gene Signature Score in SSc and normal skin

FIG. 16b Signature Score of 9-gene TUAD signature by SSc and normal skinin GSE9285 with results of the one-sided t-test comparing 9-geneSignature Score in SSc and normal skin

FIG. 17a Signature Score of 19-gene fibrosis/SSc signature by early IPF,advanced IPF and healthy lung in GSE24206 with results of the one-sidedt-test comparing 19-gene Signature Score in early IPF and normal lungand advanced IPF and normal lung, respectively

FIG. 17b Signature Score of 9-gene TUAD signature by early IPF, advancedIPF and healthy lung in GSE24206 with results of the one-sided t-testcomparing 9-gene Signature Score in early IPF and normal lung andadvanced IPF and normal lung, respectively

FIG. 18a Signature Score of 19-gene fibrosis/SSc signature by IPAH(PPH), IPF, SSc-PAH, SSc-PF and normal lung (NL) in GSE48149 withresults of the one-sided t-test comparing 19-gene Signature Score in IPFand normal lung and SSc-PF and normal lung, respectively

FIG. 18b Signature Score of 9-gene TUAD signature by IPAH (PPH), IPF,SSc-PAH, SSc-PF and normal lung in GSE48149 with results of theone-sided t-test comparing 9-gene Signature Score in IPF and normal lungand SSc-PF and normal lung, respectively

FIG. 19a Signature Score of 19-gene SSc/fibrosis signature by Nash),steatosis, healthy obese and normal liver in GSE48452 with results ofthe one-sided t-test comparing 19-gene Signature Score in Nash,steatosis and heathy obese against control liver tissue, respectively

FIG. 19b Signature Score of 9-gene TUAD signature by Nash, steatosis,heathy obese and normal liver in GSE48452 with results of the one-sidedt-test comparing 9-gene Signature Score in Nash, steatosis and heathyobese against control liver tissue, respectively

FIG. 20a Signature Score of 19-gene SSc/fibrosis signature mild andadvanced stage liver fibrosis in GSE49541 with results of the one-sidedt-test

FIG. 20b Signature Score of 9-gene TUAD signature mild and advancedstage liver fibrosis in GSE49541 with results of the one-sided t-test

FIG. 21a Signature Score of 19-gene SSc/fibrosis signature by Nash(Non-alcoholic fatty liver disease), PBC (primary biliary cholangitis),NAFLD (Non-alcoholic fatty liver disease), healthy obese and normalliver in GSE61260 with results of the one-sided t-test comparing 19-geneSignature Score in Nash, PSC, PBC and NAFLD against control livertissue, respectively

FIG. 21b Signature Score of 9-gene TUAD signature by Nash (Non-alcoholicfatty liver disease), PSC (Primary sclerosing cholangitis), PBC (primarybiliary cholangitis), NAFLD (Non-alcoholic fatty liver disease), healthyobese and normal liver in GSE61260 with results of the one-sided t-testcomparing 9-gene Signature Score in Nash, PSC, PBC and NAFLD againstcontrol liver tissue, respectively

FIG. 22a Signature Score of 19-gene SSc/fibrosis signature by primarymyelofibrosis and normal bone marrow in GSE44426 with the results of theone-sided t-test

FIG. 22b Signature Score of 9-gene TUAD signature by primarymyelofibrosis and normal bone marrow in GSE44426 with the results of theone-sided t-test

DETAILED DESCRIPTION OF THE INVENTION

The known monoclonal anti-alpha v antibody DI17E6 (designated hereinalso as Abituzumab, abituzumab, EMR62242 or EMD 525797) is found to behighly effective in interfering with cell signalling processes relevantfor the development, occurrence and/or manifestation of fibrosis andespecially of fibrotic disorders.

Without being bound by the mechanisms discussed in detail above and/orbelow and especially discussed below, it is strongly believed andsufficiently evidenced in the examples and data contained herein thatdue to the unique combination of the targeted site, the bindingaffinities/binding properties, and selectivity profile of antibodyDI17E6, and preferably its biologically active variants or modificationsthereof as discussed herein, the interaction of the antibody DI17E6, andpreferably also its biologically active variants or modificationsthereof as discussed herein, with the signalling pathways is crucial inthe development of fibrosis and especially the pathways crucial fortreating fibrosis and/or fibrotic disorders, especially the fibroticdisorders as described herein. In the light of our understanding of theresults and data underlying the instant invention, the relevant pathwaysare discussed in more detail below.

In general, fibrotic diseases are characterized by excessive scarringdue to production, deposition and contraction of extracellular matrix,which is believed to be driven by myofibroblast proliferation andactivation. Fibrotic diseases represent one of the largest groups ofdiseases for which there is no effective therapy to date. The fibroticprocesses are regulated by complex set of interactions within a networkof profibrotic and antifibrotic mediators. TGF-β (i.e. Tranforming GrothFactor beta, often also referred to as TGFb, TGF b, TGFB, TGF B, TGF-b,TGF-B, TGFbeta, TGF beta or TGF-beta) signaling is believed to play animportant role in fibroblast to myofibroblast transition (FMT) whichcontributes to increased extracellular matrix deposition, and thus isbelieved to be a main driver of disease.

TGF-β isoforms are synthesized as latent precursors complexed withlatent TGF-β binding proteins, which contains a Latency AssociatedPeptide (LAP) region. There is substantial evidence for crosstalkbetween αv integrins and TGF-β during these processes. The LAP of TGF-β1contains an RGD motif which interacts with the integrins αvβ1, αvβ3,αvβ5, αvβ6 and αvβ8 resulting in activation of TGF-β1. Abituzumab is apan-αv integrin antibody that was found to allosterically to theligand-binding αv subunit and thus prevents ligand from binding to allαvβ heterodimers and therefore inhibits αv integrin-dependent activationof latent TGF-β and thus blocks acquisition of the myofibroblastphenotype by fibroblasts and other precursors.

The obtained data demonstrate that the monoclonal anti-alpha αv antibodyDI17E6 and/or a biologically active variant or modification thereof iscapable of blocking multiple functions of αv integrins, includingbinding to RGD containing sequences in αv-integrin ligands, such asvitronectin, fibronectin and the latency associated protein of TGF-β1(LAP-β1).

Thus, one of the prominent functions of αv integrins is found to be thecontrol of the activation of TGF-β. Therefore, based on the datadiscussed herein, it is believed that cytokine TGF-β is the mainregulator of physiologic fibrogenesis and pathologic fibrosis, includingSSc as described herein, and that the anti-αv integrin antibody DI17E6,or a biologically active variant or modification thereof, is able tocontrol the activation of TGF-β in a manner that appears to beadvantageous for the treatment of fibrosis, fibrotic diseases and/orsystemic sclerosis.

Aside from this role, TGF-β has many other functions in tissue repair,angiogenesis, immunoregulation, and cell proliferation anddifferentiation. TGF-β can be secreted by platelets,monocytes/macrophages, T cells, and fibroblasts. Its signalling and cellregulation is highly complex.

Most TGF-β producing cells generate it as a biologically inactiveprecursor molecule that resides as a latent complex in the ECM reservoirand is unable to interact with its receptors. The conversion of latentTGF-β to its active form capable of binding its cell surface receptorsis mediated by molecules such as thrombospondin-1, certain αvβx integrinheterodimers (FIG. 1), and various proteases, and is tightly regulated.Activated TGF-β binds to the type II TGF-β receptor, triggering of anintracellular signal transduction cascade that leads to induction oftarget genes. So far there is evidence for αvβ3, αvβ3, αvβ6 and αvβ8 cancontrol the activation of TGF-β. This is described and discussed in moredetail above and/or below.

The cytokine TGF-β is considered to be the main regulator of physiologicfibrogenesis and pathologic fibrosis, including SSc (see also thesection relating to “Histopathological and pathophysiologicalcharacteristics”). The numerous cellular effects of TGF-β are describedherein, and some of the most pertinent roles of TGF-β, including itsconnection and/or its association with integrins, are given in Table 7(below).

TABLE 7 Fibrogenic activities of TGF-β Recruits monocytes Stimulatessynthesis of collagens, fibronectin, proteoglycans, elastin, tissueinhibitor of metalloproteinases, inhibits matrix metalloproteinasesStimulates fibroblast proliferation, chemotaxis Induces fibrogeniccytokine production (CTGF), autoinduction, blocks synthesis and activityof interferon-gamma (IFN-γ) Stimulates production of endothelin-1Stimulates expression of surface receptors for TGF-β, PDGF Inducesfibroblast mitogenic responses to PDGF-AA Promotesfibroblast-myofibroblast differentiation, monocyte-fibrocytedifferentiation Promotes epithelial-to mesenchymal transition (EMT)Inhibits fibroblast apoptosis Induces expression of αv integrins

Thus, a preferred subject of the instant invention relates to theanti-αv integrin antibody DI17E6, or a biologically active variant ormodification thereof, for use in the treatment of patients sufferingfrom fibrotic diseases and especially systemic sclerosis (SSc).Preferably, the terms “fibrotic diseases” and/or “systemic sclerosis”have the meaning and characteristics as is known in the art. Morepreferably, the terms fibrotic diseases”, and/or “systemic sclerosis”have the meanings and characteristics as described above and/or below.

Thus, a preferred subject of the instant invention relates to theanti-αv integrin antibody DI17E6, or a biologically active variant ormodification thereof, for use in systemic sclerosis, wherein thesystemic sclerosis comprises systemic sclerosis of the lung, liver,kidney, cardiovascular system and/or or skin. More preferably, thedisease to be treated according to the invention is selected fromsystemic sclerosis of the lung, the liver and the kidney. Especiallypreferably, the disease to be treated according to the invention is thesystemic sclerosis of the lung or comprises the systemic sclerosis ofthe lung.

Likewise preferred is the anti-αv integrin antibody DI17E6, or abiologically active variant or modification thereof, for use in systemicsclerosis, preferably for use in systemic sclerosis as described aboveand/or below in more detail, preferably wherein the systemic sclerosisaffects one or more organs selected from the group consisting of lung,liver, kidney, heart and skin, more preferably lung, liver, kidneyand/or heart, and especially lung or heart.

Also likewise preferred is the anti-αv integrin antibody DI17E6, or abiologically active variant or modification thereof, for use in thetreatment of systemic sclerosis, preferably for use in the treatment ofsystemic sclerosis as described above and/or below in more detail,wherein the systemic sclerosis affects the cardiovascular system, theblood vessels and/or the blood. Thus, the disease to be treatedaccording to the invention is preferably selected from diastolicdysfunction and myelofibrosis.

Thus, even more preferred is the anti-αv integrin antibody DI17E6, or abiologically active variant or modification thereof, for use, preferablyfor use as described in more detail above and/or below, wherein thesystemic sclerosis comprises one or more indications selected from thegroup consisting of idiopathic pulmonary fibrosis, primary sclerosingcholangitis, non-alcoholic steatohepatitis (NASH), primary focalglomerulosclerosis, primary segmental glomerulosclerosis, diabeticnephropathy, diastolic dysfunction and myelofibrosis.

Thus, even more preferred is the anti-αv integrin antibody DI17E6, or abiologically active variant or modification thereof, for use, preferablyfor use as described in more detail above and/or below, wherein thesystemic sclerosis comprises an indication or disease, wherein one ormore of the clinical pictures or manifestations of both focalglomerulosclerosis, or primary focal glomerulosclerosis, and segmentalglomerulosclerosis, or primary focal glomerulosclerosis, are present.Accordingly, even more preferred is the anti-αv integrin antibodyDI17E6, or a biologically active variant or modification thereof, foruse, preferably for use as described in more detail above and/or below,in the treatment of focal segmental glomerulosclerosis (FSGS).

Especially preferred is thus the anti-αv integrin antibody DI17E6, or abiologically active variant or modification thereof, for use, preferablyfor use as described in more detail above and/or below, wherein saidtreatment comprises patients suffering from pulmonary fibrosis and/oralveolitis (interstitial lung disease, ILD).

Alternatively preferred is the anti-αv integrin antibody DI17E6, or abiologically active variant or modification thereof, for use accordingto claim 1, wherein the disease to be treated is systemic sclerosis ofthe skin.

Preferably, the systemic sclerosis of the skin is selected from thegroup consisting of diffuse cutaneous systemic sclerosis (dcSSc) andlimited cutaneous systemic sclerosis (IcSSc).

Especially preferred subjects of the invention include:

The anti-αv integrin antibody DI17E6 or a biologically active variant ormodification thereof, preferably the anti-αv integrin antibody DI17E6,for use in the treatment of pulmonary fibrosis, alveolitis (interstitiallung disease, ILD), and/or sclerodermal interstitial lung disease(SSc-ILD).

The anti-αv integrin antibody DI17E6 or a biologically active variant ormodification thereof, preferably the anti-αv integrin antibody DI17E6,for use in the treatment as described above and/or below, wherein saidtreatment comprises the administration of a dose, preferably aneffective dose, of said antibody, or biologically active variant ormodification thereof, in an amount of 10 mg-1000 mg per week or per twoweeks.

The anti-αv integrin antibody DI17E6 or a biologically active variant ormodification thereof, preferably the anti-αv integrin antibody DI17E6,for use in the treatment as described above and/or below, wherein saidtreatment comprises the administration of a dose, preferably aneffective dose, of said antibody, or biologically active variant ormodification thereof, in an amount of about 500 mg, about 1000 mg orabout 1500 mg within 4 weeks or within a month. Preferably, theadministration of said dose is repeated several times every 4 weeks orevery month, respectively.

The anti-αv integrin antibody DI17E6 or a biologically active variant ormodification thereof, preferably the anti-αv integrin antibody DI17E6,for use in the treatment as described above and/or below, wherein saidtreatment comprises the administration of a dose, preferably aneffective dose, of said antibody, or biologically active variant ormodification thereof, in an amount of about 500 mg every 4 weeks.

The anti-αv integrin antibody DI17E6 or a biologically active variant ormodification thereof, preferably the anti-αv integrin antibody DI17E6,for use in the treatment as described above and/or below, wherein saidtreatment comprises the administration of a dose, preferably aneffective dose, of said antibody, or biologically active variant ormodification thereof, in an amount of about 1000 mg every 4 weeks.

The anti-αv integrin antibody DI17E6 or a biologically active variant ormodification thereof, preferably the anti-αv integrin antibody DI17E6,for use in the treatment as described above and/or below, wherein saidtreatment comprises the administration of a dose, preferably aneffective dose, of said antibody, or biologically active variant ormodification thereof, in an amount of about 1500 mg every 4 weeks.

Preferably, said administration of said dose every 4 weeks or everymonth, respectively, is repeated at least 4 times, more preferably atleast 8 times, even more preferably at least 16 times and especially atleast 24 times.

Typically, said administration of said dose every 4 weeks or every monthis repeated for about one year, for about one and a half year, for about2 years, or for about two and a half or for about three years.

Thus, said administration of said dose every 4 weeks or every month,respectively, is preferably repeated not more than about 36 times, morepreferably not more than about 28 times, even more preferably not morethan about 24 times and especially not more than about 16 times or about12 times.

Accordingly, preferred ranges for the duration of said repeatedadministrations are 4 to 36 months, 8 to 36 months, 12 to 36 months, 8to 28 months, 12 to 28 months, or 16 to 28 months.

However, in principle, there is no upper limit for said repeatedadministration. However, it may be reasonable to stop said repeatedadministration, at least temporarily, after about half a year, afterabout one year, after about one and a half year, after about 2 years orafter about two and a half years, e.g. in order to see how the patient'sstate evolved and to decide whether or not a new repeated administrationshall be started. The above described repeated administration isespecially preferred with regard to Abituzumab.

The anti-αv integrin antibody DI17E6 or a biologically active variant ormodification thereof, preferably the anti-αv integrin antibody DI17E6,for use in the treatment as described above and/or below, and preferablyfor use as described in the paragraph directly above wherein the dose,preferably the effective dose, is administered in a single dose.

The anti-αv integrin antibody DI17E6 or a biologically active variant ormodification thereof, preferably the anti-αv integrin antibody DI17E6,for use in the treatment as described above and/or below, and preferablyfor use as described in at least one of the two paragraphs directlyabove, wherein said antibody or said biologically active variant ormodification thereof is administered as monotherapy.

The anti-αv integrin antibody DI17E6 or a biologically active variant ormodification thereof, preferably the anti-αv integrin antibody DI17E6,for use as described above and/or below, wherein said biological activevariant or modification comprises the CDR regions and heavy and lightchain variable regions of DI17E6, which are at least 80% identical inamino acid sequence compared to the variable regions of DI17E6,preferably at least 90% identical in amino acid sequence compared to thevariable regions of DI17E6, more preferably at least 95% identical inamino acid sequence compared to the variable regions of DI17E6, evenmore preferably at least 98% identical in amino acid sequence comparedto the variable regions of DI17E6, and especially at least 99% identicalin amino acid sequence compared to the variable regions of DI17E6.

The DI17E6 antibody for use as described above and/or below, andespecially as described in the paragraph directly above, comprising oneor more modifications within the heavy chain framework regions

FR1: (SEQ ID No. 16) QVQLQQSG A ELA E PGASVK M SCKASGYTFS FR2:(SEQ ID No. 17) WV K Q R PGQGLEWIG FR3: (SEQ ID No. 18) KATMT A DTS SSTAYM Q LS G L T SED S AVYYCAS FR4: (SEQ ID No. 19) WGQGT S VTVSS,

-   -   wherein one or more of the bold and underlined positions are        mutated and are different compared to the original respective        sequence.

The DI17E6 antibody and/or or a biologically active variant ormodification thereof for use as described above and/or below, whereinthe biological active variant or modification comprises a constantregion, which is at least 80% identical in amino acid sequence comparedto the constant region of DI17E6, preferably which is at least 90%identical in amino acid sequence compared to the constant region ofDI17E6, more preferably which is at least 95% identical in amino acidsequence compared to the constant region of DI17E6, even more preferablywhich is at least 98% identical in amino acid sequence compared to theconstant region of DI17E6, and especially which is which is at least 99%identical in amino acid sequence compared to the constant region ofDI17E6.

The DI17E6 antibody and/or or a biologically active variant ormodification thereof for use as described above and/or below, comprisinga human IgG1 constant region instead of human IgG2, or a human IgG2hinge region instead of the human IgG1 hinge.

Further especially preferred subjects of the instant invention include:

A method of treating fibrotic diseases, preferably systemic sclerosisand especially systemic sclerosis as described above and/or below,comprising administering to a patient the DI17E6 antibody and/or abiologically active variant or modification thereof, wherein thebiologically active variant or modification comprises the CDR regionsand heavy and light chain variable regions, which are 80%-95% identicalin amino acid sequence compared to the variable regions of DI17E6.

A method of treating fibrotic diseases, preferably systemic sclerosisand especially systemic sclerosis as described above and/or below,comprising administering to a patient the DI17E6 antibody and/or abiologically active variant or modification thereof, wherein thebiological active variant or modification comprises a constant region,which is at least 80%-98% identical with the amino acid sequencecompared to the constant region of DI17E6.

A method of treating fibrotic diseases, preferably systemic sclerosisand especially systemic sclerosis as described above and/or below,comprising administering to a patient the DI17E6 antibody and/or abiologically active variant or modification thereof, comprising one ormore modifications within the heavy chain framework regions

FR1: (SEQ ID No. 16) QVQLQQSG A ELA E PGASVK M SCKASGYTFS FR2:(SEQ ID No. 17) WV K Q R PGQGLEWIG FR3: (SEQ ID No. 18) KATMT A DTS SSTAYM Q LS G L T SED S AVYYCAS FR4: (SEQ ID No. 19) WGQGT S VTVSS,

-   -   wherein one or more of the bold and underlined positions are        mutated and are different compared to the original respective        sequence.

The respective method as described above and/or below, preferably asdescribed directly above, comprising the administration of a modifiedDI17E6 antibody comprising a human IgG1 constant region instead of humanIgG2, or a human IgG2 hinge region instead of the human IgG1 hingeregion.

The safety results of the phase 1, open-label study showed that repeatedinfusions of single-agent DI17E6 (EMD 525797) at each of four doselevels are generally well tolerated and appear to be safe in patients.There are no dose-limiting toxicities (DLT) and no infusion reactions.With regard to dose, no trends in the distribution of TEAEs, NCI-CTCAE(version 3.0) grade or drug relationship are observed. In addition,there is no evidence of accumulation of any specific event withinindividual cohorts. Eleven patients experienced TEAEs that areconsidered to be drug-related. In this regard, skin symptoms such aspruritus, erythema and rash, which are reported in a total of fourpatients, are predictable adverse events associated with DI17E6 (EMD525797) given that integrins are responsible for the maintenance of theepithelial phenotype. Symptoms of mucosal inflammation and swollentongue may also be characteristic of the mechanism of action of EMD525797, but together with fatigue, might also be signs of the underlyingdisease. The hematologic and biochemic toxicity shifts observed in eightpatients could also be explained by underlying disease, as well asconcomitant medications.

PK assessment after single and multiple doses of study drug suggest thatDI17E6 (EMD 525797) behaved in accordance with a receptor-mediatedclearance model as described for other antibodies targetingmembrane-associated receptors. Consistent with the findings of anearlier study in healthy volunteers, PKs of DI17E6 (EMD 525797) in mCRPCpatients are dose-dependent with clearance determined predominantly bythe availability of unbound receptors. At the doses used in the presentstudy, it can be assumed that at doses of 1000 mg or higher, almost allreceptors are saturated and have a minor contribution to drug clearance.Immunologically triggered antibodies directed against DI17E6 can bedetected in some (16%) patients; however, no impact on PKs or safetycould be found.

In conclusion, single-agent EMD 525797 given as single and multipledoses is shown to be well tolerated in patients. No safety concern canbe identified and there is preliminary evidence of clinical benefit innumerous patients. Due to its target and safety profile, DI17E6 (EMD525797) is a promising agent for single agent and/or combinationtherapy.

Thus, preferred subjects of the instant invention thus are:

-   -   The DI17E6 antibody as described above and/or below for use in        the treatment of disorders as described above and/or below,        wherein the dose, preferably the effective dose, of the antibody        is 500 mg-1500 mg per two weeks or 1000-3000 mg per month,        preferably 500-1000 mg per two weeks or 1000-2000 mg per month.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of disorders as described above and/or below,        wherein the dose, preferably the effective dose, of the antibody        is about 500 mg per month, about 1000 mg per month, about 1500        mg per month or about 2000 mg per month.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of disorders as described above and/or below,        wherein the dose, preferably the effective dose, of 500-1000 mg        is administered by a single infusion.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of disorders as described above and/or below,        wherein the dose, preferably the effective dose, of 1000-2000 mg        is administered by a single infusion.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of disorders as described above and/or below,        wherein the dose, preferably the effective dose, of 500-1000 mg        is administered by a single infusion once a month.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of disorders as described above and/or below,        wherein the dose, preferably the effective dose, of 1000-2000 mg        is administered by a single infusion once a month.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of disorders as described above and/or below,        wherein the dose, preferably the effective dose, of about 500 mg        is administered by a single infusion.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of disorders as described above and/or below,        wherein the dose, preferably the effective dose, of about 1000        mg is administered by a single infusion.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of disorders as described above and/or below,        wherein the dose, preferably the effective dose, of about 1500        mg is administered by a single infusion.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of disorders as described above and/or below,        wherein the dose, preferably the effective dose, of about 1500        mg is administered by a single infusion once a month.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of disorders as described above and/or below,        wherein the dose, preferably the effective dose, of about 1500        mg is administered by a single infusion once every 4 weeks.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of disorders as described above and/or below,        wherein the antibody is administered in a monotherapy setting        without additional cotherapeutic agents.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of disorders as described above and/or below,        wherein the antibody is administered in an combination therapy        setting in combination with additional cotherapeutic agents.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of disorders as described above and/or below,        wherein the antibody is administered in an combination therapy        setting in combination with MMF (Mycophenolat or Mycophenolate).    -   The DI17E6 antibody as described above and/or below for use in        the treatment of patients with SSc-ILD, wherein the antibody is        administered in a combination therapy setting in combination        with MMF (Mycophenolat or Mycophenlate).    -   The DI17E6 antibody as described above and/or below for use in        the treatment of patients with SSc-ILD, wherein the antibody is        administered in an amount of about 500 mg per month, preferably        in an amount of about 500 mg as a single administration once a        month, in an combination therapy setting in combination with MMF        (Mycophenolat or Mycophenolate).    -   The DI17E6 antibody as described above and/or below for use in        the treatment of patients with SSc-ILD, wherein the antibody is        administered in an amount of about 1.000 mg per month,        preferably in an amount of about 1.000 mg as a single        administration once a month, in an combination therapy setting        in combination with MMF (Mycophenolat or Mycophenolate).    -   The DI17E6 antibody as described above and/or below for use in        the treatment of patients with SSc-ILD, wherein the antibody is        administered in an amount of about 1.500 mg per month,        preferably in an amount of about 1.500 mg as a single        administration once a month, in an combination therapy setting        in combination with MMF (Mycophenolat or Mycophenolate).    -   The DI17E6 antibody as described above and/or below for use in        the treatment of fibrosis, preferably excessive and/or        pathological fibrosis, preferably in a manner as described above        and/or below.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of fibrotic disorders, preferably fibrotic        disorders as described above and/or below, preferably in a        manner as described above and/or below.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of systemic sclerosis (SSc), preferably in a        manner as described above and/or below.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of disorders as described above and/or below,        wherein the organs affected by said disorders are selected from        the group consisting of lung, liver, kidney, cardiovascular        system or skin.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of fibrosis, preferably excessive and/or        pathological fibrosis, preferably in a manner as described above        and/or below, wherein the fibrosis, excessive fibrosis and/or        pathological fibrosis affects one or more organs selected from        the group consisting of lung, liver, kidney, heart and skin.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of fibrotic disorders, preferably fibrotic        disorders as described above and/or below, preferably in a        manner as described above and/or below, wherein said fibrotic        disorder affects one or more organs selected from the group        consisting of lung, liver, kidney, heart and skin.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of systemic sclerosis (SSc), wherein the systemic        sclerosis comprises systemic sclerosis of the lung, liver,        kidney, cardiovascular system and/or skin.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of systemic sclerosis (SSc), wherein said systemic        sclerosis affects one or more organs selected from the group        consisting of lung, liver, kidney, heart and skin.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of systemic sclerosis (SSc), wherein the systemic        sclerosis affects the cardiovascular system, the blood vessels        and/or the blood.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of systemic sclerosis (SSc), wherein said systemic        sclerosis affects the lung and/or the skin.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of systemic sclerosis (SSc), wherein said systemic        sclerosis affects the lung.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of systemic sclerosis (SSc), wherein said systemic        sclerosis affects the skin.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of systemic sclerosis (SSc), wherein said systemic        sclerosis affects the liver.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of systemic sclerosis (SSc), wherein said systemic        sclerosis affects the kidneys.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of systemic sclerosis (SSc), wherein said systemic        sclerosis affects the heart.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of systemic sclerosis (SSc), wherein the systemic        sclerosis comprises one or more indications selected from the        group consisting of idiopathic pulmonary fibrosis, primary        sclerosing cholangitis, non-alcoholic steatohepatitis (NASH),        primary focal glomerulosclerosis, primary segmental        glomerulosclerosis, diabetic nephropathy, diastolic dysfunction        and myelofibrosis.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of systemic sclerosis (SSc), wherein said systemic        sclerosis comprises pulmonary fibrosis and/or alveolitis        (interstitial lung disease, ILD).    -   The DI17E6 antibody as described above and/or below for use in        the treatment of pulmonary fibrosis and/or alveolitis        (interstitial lung disease, ILD).    -   The DI17E6 antibody as described above and/or below for use in        the treatment of SSc-ILD or scleroderma-ILD.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of patients suffering from SSc-ILD.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of patients suffering from scleroderma-ILD.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of cutaneous systemic sclerosis (dcSSc) or limited        cutaneous systemic sclerosis (IcSSc).    -   The DI17E6 antibody as described above and/or below for use in        the treatment of subjects, preferably human subjects, with        systemic sclerosis-associated interstitial lung disease        (SSc-ILD).    -   Abituzumab for use in the treatment of subjects, preferably        human subjects, with systemic sclerosis-associated interstitial        lung disease (SSc-ILD).    -   The DI17E6 antibody as described above and/or below for use in        the treatment of subjects, preferably human subjects, with        SSc-ILD who already receive mycophenolate.    -   The DI17E6 antibody as described above and/or below for use in        the treatment of subjects, preferably human subjects, with        SSc-ILD who already receive mycophenolate, preferably constant        doses of mycophenolate.    -   Abituzumab for use in the treatment of subjects, preferably        human subjects, with SSc-ILD who already receive mycophenolate.    -   The biologically active variant or modification of said anti-αv        integrin antibody DI17E6 as described above and/or below for use        in the treatment of fibrosis and/or fibrotic disorders, wherein        said biological active variant or modification comprises the CDR        regions and heavy and light chain variable regions of DI17E6,        which are at least 80% identical, at least 90% identical, at        least 95% identical, at least 98% identical or at least 99%        identical in amino acid sequence compared to the variable        regions of DI17E6.    -   The biologically active variant or modification of said anti-αv        integrin antibody DI17E6 as described above and/or below for use        in the treatment of fibrosis and/or fibrotic disorders, wherein        said biological active variant or modification comprises the        heavy and/or light chain variable regions of DI17E6, which are        at least 90% identical, at least 95% identical, at least 98%        identical, at least 99% identical, or at least 99.5% identical        in amino acid sequence compared to the respective heavy and/or        light chain variable regions of DI17E6.    -   The biologically active variant or modification of said anti-αv        integrin antibody DI17E6 as described above and/or below for use        in the treatment of fibrosis and/or fibrotic disorders, wherein        said biological active variant or modification comprises the        heavy and/or light chain CDR regions of DI17E6, which are at        least 90% identical, at least 92% identical, at least 94%        identical, at least 96% identical, at least 98% or at least 99%        identical in amino acid sequence compared to the respective        heavy and/or light chain CDR regions of DI17E6.    -   The biologically active variant or modification of said anti-αv        integrin antibody DI17E6 as described above and/or below,        comprising one or more modifications within the heavy chain        framework regions

FR1: (SEQ ID No. 16) QVQLQQSG A ELA E PGASVK M SCKASGYTFS FR2:(SEQ ID No. 17) WV K Q R PGQGLEWIG FR3: (SEQ ID No. 18) KATMT A DTS SSTAYM Q LS G L T SED S AVYYCAS FR4: (SEQ ID No. 19) WGQGT S VTVSS,

-   -    wherein one or more of the bold and underlined positions are        mutated and are different compared to the original respective        sequence of DI17E6, for use in the treatment of fibrosis and/or        fibrotic disorders as described above and/or below.    -   The biologically active variant or modification of said anti-αv        integrin antibody DI17E6 as described above and/or below for use        in the treatment of fibrotic disorders, preferably fibrotic        disorders as described above and/or below, preferably in a        manner as described above and/or below, wherein said fibrotic        disorder affects one or more organs selected from the group        consisting of lung, liver, kidney, heart and skin.    -   The biologically active variant or modification of said anti-αv        integrin antibody DI17E6 as described above and/or below for use        in the treatment of systemic sclerosis (SSc), wherein the        systemic sclerosis comprises systemic sclerosis of the lung,        liver, kidney, cardiovascular system and/or skin.    -   The biologically active variant or modification of said anti-αv        integrin antibody DI17E6 as described above and/or below for use        in the treatment of systemic sclerosis (SSc), wherein said        systemic sclerosis affects one or more organs selected from the        group consisting of lung, liver, kidney, heart and skin.    -   The biologically active variant or modification of said anti-αv        integrin antibody DI17E6 as described above and/or below for use        in the treatment of systemic sclerosis (SSc), wherein the        systemic sclerosis affects the cardiovascular system, the blood        vessels and/or the blood.    -   The biologically active variant or modification of said anti-αv        integrin antibody DI17E6 as described above and/or below for use        in the treatment of systemic sclerosis (SSc), wherein said        systemic sclerosis affects the lung and/or the skin.    -   The biologically active variant or modification of said anti-αv        integrin antibody DI17E6 as described above and/or below for use        in the treatment of systemic sclerosis (SSc), wherein the        systemic sclerosis comprises one or more indications selected        from the group consisting of idiopathic pulmonary fibrosis,        primary sclerosing cholangitis, non-alcoholic steatohepatitis        (NASH or Nash), primary focal glomerulosclerosis, primary        segmental glomerulosclerosis, diabetic nephropathy, diastolic        dysfunction and myelofibrosis.    -   The biologically active variant or modification of said anti-αv        integrin antibody DI17E6 as described above and/or below for use        in the treatment of systemic sclerosis (SSc), wherein said        systemic sclerosis comprises pulmonary fibrosis and/or        alveolitis (interstitial lung disease, ILD).    -   The biologically active variant or modification of said anti-αv        integrin antibody DI17E6 as described above and/or below for use        in the treatment of pulmonary fibrosis and/or alveolitis        (interstitial lung disease, ILD).    -   The biologically active variant or modification of said anti-αv        integrin antibody DI17E6 as described above and/or below for use        in the treatment of SSc-ILD or scleroderma-ILD.    -   A method of treating fibrosis and/or fibrotic diseases in a        subject by administering the anti-αv integrin antibody DI17E6,        or a biologically active variant or modification thereof,        wherein said subject is characterized by a higher-than-threshold        score calculated from a multi-gene signature comprising 2 or        more, preferably 5 or more, more preferably 9 or more, even more        preferably 15 or more and especially 16, 17, 18 or 19 genes,        selected from the group consisting of the genes COL15A1        (NM_001855), COL1A1 (NM_000088), COMP (NM_000095), RGS5        (NM_003617), COL10A1 (NM_000493), COL5A1 (NM_000093), IGFBP2        (NM_000597), NM_005576, MOXD1 (NM_015529), ADRA2A (NM_000681),        COL5A2 (NM_000393), MMP10 (NM_002425), TNFRSF21 (NM_014452),        ITGA7 (NM_002206), TGF-β3 (NM_003239), MMP11 (NM_005940), SPP1        (NM_000582), CCL2 (NM_002982), and TNC (NM_002160), in        particular the 9-gene signature based on the genes COL15A1        (NM_001855), COL1A1 (NM_000088), COMP (NM_000095), COL10A1        (NM_000493), COL5A1 (NM_000093), COL5A2 (NM_000393), ITGA7        (NM_002206), MMP11 (NM_005940), and TNC (NM_002160) hereafter        also referred to as TUAD signature.    -   A method of monitoring the severity of fibrosis in fibrotic        diseases and/or the emergence of fibrosis in diseases with        potential to develop fibrosis in a subject by tracking the        changes of a score calculated from a multi-gene signature        comprising 2 or more, preferably 5 or more, more preferably 9 or        more, even more preferably 15 or more and especially 16, 17, 18        or 19 genes, selected from the group consisting of the genes        COL15A1 (NM_001855), COL1A1 (NM_000088), COMP (NM_000095), RGS5        (NM_003617), COL10A1 (NM_000493), COL5A1 (NM_000093), IGFBP2        (NM_000597), NM_005576, MOXD1 (NM_015529), ADRA2A (NM_000681),        COL5A2 (NM_000393), MMP10 (NM_002425), TNFRSF21 (NM_014452),        ITGA7 (NM_002206), TGF-β3 (NM_003239), MMP11 (NM_005940), SPP1        (NM_000582), CCL2 (NM_002982), and TNC (NM_002160), in        particular the 9-gene signature based on the genes COL15A1        (NM_001855), COL1A1 (NM_000088), COMP (NM_000095), COL10A1        (NM_000493), COL5A1 (NM_000093), COL5A2 (NM_000393), ITGA7        (NM_002206), MMP11 (NM_005940), and TNC (NM_002160) hereafter        also referred to as TUAD signature.    -   A method of treating fibrosis and/or fibrotic diseases in a        subject by administering the anti-αv integrin antibody DI17E6,        or a biologically active variant or modification thereof,        wherein said subject is characterized by a gene signature        comprising 2 or more, preferably 5 or more, more preferably 10        or more, even more preferably 15 or more and especially 16, 17,        18 or 19 genes, selected from the group consisting of the genes        COL15A1 (NM_001855), COL1A1 (NM_000088), COMP (NM_000095), RGS5        (NM_003617), COL10A1 (NM_000493), COL5A1 (NM_000093), IGFBP2        (NM_000597), NM_005576, MOXD1 (NM_015529), ADRA2A (NM_000681),        COL5A2 (NM_000393), MMP10 (NM_002425), TNFRSF21 (NM_014452),        ITGA7 (NM_002206), TGF-β3 (NM_003239), MMP11 (NM_005940), SPP1        (NM_000582), CCL2 (NM_002982), and TNC (NM_002160), in        particular the 9-gene signature based on the genes COL15A1        (NM_001855), COL1A1 (NM_000088), COMP (NM_000095), COL10A1        (NM_000493), COL5A1 (NM_000093), COL5A2 (NM_000393), ITGA7        (NM_002206), MMP11 (NM_005940), and TNC (NM_002160) hereafter        also referred to as TUAD signature.    -   Use of DI17E6, or a biologically active variant or modification        thereof, preferably a biologically active variant or        modification thereof according as described herein, and        especially abituzumab, for the manufacture of a medicament for        the prophylaxis and/or treatment of fibrosis and/or fibrotic        disorders.    -   Use of DI17E6, or a biologically active variant or modification        thereof, preferably a biologically active variant or        modification thereof according as described herein, and        especially abituzumab, for the manufacture of a medicament for        the prophylaxis and/or treatment of fibrosis and/or fibrotic        disorders as described herein, and especially the prophylaxis        and/or treatment of one or more indications selected from the        group consisting of idiopathic pulmonary fibrosis, primary        sclerosing cholangitis, non-alcoholic steatohepatitis (NASH),        primary focal glomerulosclerosis, primary segmental        glomerulosclerosis, diabetic nephropathy, diastolic dysfunction        and myelofibrosis.    -   Use of abituzumab for the manufacture of a medicament for the        prophylaxis and/or treatment of fibrosis and/or fibrotic        disorders as described herein, and especially the prophylaxis        and/or treatment of one or more indications selected from the        group consisting of idiopathic pulmonary fibrosis, primary        sclerosing cholangitis, non-alcoholic steatohepatitis (NASH),        primary focal glomerulosclerosis, primary segmental        glomerulosclerosis, diabetic nephropathy, diastolic dysfunction        and myelofibrosis.    -   Use of abituzumab for the manufacture of a medicament for the        prophylaxis and/or treatment of systemic sclerosis, preferably        comprising pulmonary fibrosis, alveolitis (interstitial lung        disease, ILD), and/or sclerodermal interstitial lung disease        (SSc-ILD).    -   Use of abituzumab for the manufacture of a medicament for the        prophylaxis and/or treatment of focal segmental        glomerulosclerosis (FSGS).    -   Use of DI17E6, or a biologically active variant or modification        thereof, preferably a biologically active variant or        modification thereof according as described herein, and        especially abituzumab, for the manufacture of a medicament for        the prophylaxis and/or treatment of fibrosis and/or fibrotic        disorders as described herein, wherein the treatment        additionally comprises the administration of one or more active        ingredients, selected from the group consisting of mycophenolic        acid, mycophenolate, mycophenolate mofetil, mycophenolate        sodium, methotrexate, amethopterin and prednisone.    -   Use of abituzumab for the manufacture of a medicament for the        prophylaxis and/or treatment of fibrosis and/or fibrotic        disorders as described herein, wherein the treatment        additionally comprises the administration of mycophenolic acid,        mycophenolate, mycophenolate mofetil and/or mycophenolate        sodium.    -   Use of abituzumab for the manufacture of a medicament for the        prophylaxis and/or treatment of fibrosis and/or fibrotic        disorders as described herein, wherein the treatment        additionally comprises the administration of methotrexate and/or        amethopterin.    -   Use of abituzumab for the manufacture of a medicament for the        prophylaxis and/or treatment of fibrosis and/or fibrotic        disorders as described herein, wherein the treatment        additionally comprises the administration of prednisone.

Due to its uniquely different mode of action, DI17E6, or a biologicallyactive variant or modification thereof, preferably a biologically activevariant or modification thereof according as described herein, andespecially abituzumab, is deemed to be applicable in combination withbasically all treatment options applied in the prophylaxis and/ortreatment of fibrosis and/or fibrotic disorders, especially fibrosisand/or fibrotic disorders as described herein.

Thus, the addition of DI17E6, or a biologically active variant ormodification thereof, preferably a biologically active variant ormodification thereof according as described herein, and especiallyabituzumab, to treatment regimen that include the administration of atypical standard medicament in the treatment of fibrosis and/or fibroticdisorders, including, but not limited to one or more active ingredients,selected from the group consisting of mycophenolic acid, mycophenolate,mycophenolate mofetil, mycophenolate sodium, methotrexate, amethopterinand prednisone.

Thus, especially preferred is the anti-αv integrin antibody DI17E6, orthe biologically active variant or modification thereof, for use asdescribed above and/or below, preferably as described above, whereinsaid antibody, or said modification thereof, is the antibody with theregistered International Non-proprietary Name (INN) Abituzumab.Likewise, especially preferred is a method as described above and/orbelow, preferably as described above, wherein said antibody, or saidmodification thereof, is the antibody with the registered InternationalNon-proprietary Name (INN) Abituzumab. Likewise, especially preferred isthe use of the anti-αv integrin antibody DI17E6, or the biologicallyactive variant or modification thereof, for the manufacture of amedicament for the prophylaxis and/or treatment of fibrosis and/orfibrotic disorders, preferably as described above and/or below, andespecially preferably as described above, wherein said antibody, or saidmodification thereof, is the antibody with the registered InternationalNon-proprietary Name (INN) Abituzumab.

According to the instant invention, the anti-αv integrin antibodyDI17E6, preferably also referred to herein as Abituzumab or abituzumab,is an engineered specifically tailored IgG2 hybrid monoclonal antibodydirected to alpha-v integrin (receptor). This antibody is described indetail in WO 2009/010290, the disclosure of which is incorporated hereinin its entirety.

Its hypervariable regions (CDRs) derive from murine mAb 17E6 (EMD73034). This parent mouse IgG1 antibody is described, for example byMitjans et al. (1995; J. Cell Sci. 108, 2825) and patents U.S. Pat. No.5,985,278 and EP 719 859. Mouse mAb 17E6 is produced by hybridoma cellline 272-17E6 and deposited under accession number DSM ACC2160.

Its light chain domains derive from humanized monoclonal anti-EGFRantibody 425 (matuzumab). This antibody is described in detail forexample in EP 0 531 472B1, and derives from its murine counterpart 425(mouse MAb 425, ATCC HB9629), The antibody was raised against the humanA431 carcinoma cell line and found to bind to a polypeptide epitope onthe external domain of the human epidermal growth factor receptor(EGFR). Matuzumab has shown in clinical trials high efficacy.

Generally, the anti-αv integrin antibody DI17E6 as used according to theinvention comprises:

-   (i) a CDR light and a heavy chain region deriving from mouse    monoclonal anti-αv integrin antibody 17E6-   (ii) a light chain framework region which is taken from humanized    monoclonal anti-EGFR antibody 425,-   (iii) a heavy chain framework region deriving from mouse monoclonal    anti-αv integrin antibody 17E6, optionally comprising one or more    mutations of amino acids at specific positions, and-   (iv) a heavy chain constant region deriving from human IgG2 and a    human constant kappa light chain region, wherein in said IgG2 domain    the IgG2 hinge region was replaced by the human IgG1 hinge domain,    and; wherein optionally one or more mutations within the IgG2 has    been carried out.

Specifically, DI17E6 (designated as “DI17E6γ2h(N297Q)” or “EMD 525797”)as used for the treatment as claimed and in the clinical trials asdescribed above and below, has the following amino acid sequence:

(i) variable and constant light chain sequences (SEQ ID No. 1):DIQMTQSPSSLSASVGDRVTITCRASQDISNYLAWYQQKPGKAPKLLIYYTSKIHSGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTFPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC and(ii) variable and constant heavy chain sequences (SEQ ID No. 2):QVQLQQSGGELAKPGASVKVSCKASGYTFSSFWMHWVRQAPGQGLEWIGYINPRSGYTEYNEIFRDKATMTTDTSTSTAYMELSSLRSEDTAVYYCASFLGRGAMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVEPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQAQSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK,wherein the underlined sequences represent the variable regions with theCDRs (in bold, identical with the parent mouse antibody). The modifiedIgG1 hinge region is represented by EPKSSDKTHTCPPCP (SEQ ID No. 3), andAQ is a substitution within the IgG2 domain.

However, as it was shown in WO 2009/010290, also variants of DI17E6 canbe used according to the teaching of this invention. Thus, DI17E6variants comprising one or more modifications within the heavy chainframework regions

FR1: (SEQ ID No. 16) QVQLQQSG A ELA E PGASVK M SCKASGYTFS FR2:(SEQ ID No. 17) WV K Q R PGQGLEWIG FR3: (SEQ ID No. 18) KATMT A DTS SSTAYM Q LS G L T SED S AVYYCAS FR4: (SEQ ID No. 19) WGQGT S VTVSS,wherein one or more of the bold and underlined positions are mutated,can be used in the treatment of prostate cancer patients as described.In more detail, the following position heavy chain framework region ismutated at one, more or all of the following positions can be mutated:A9, E13, M20, K38, R40, A72, S76, Q82, G85, T87, S91 and S113. Thesevariants show the same or very similar biological activity and efficacyas compared to DI17E6 defined by its sequences above.

In general, the invention as described includes also modifications andvariants of the DI17E6 antibody that are functionally and/orpharmaceutically identical or similar to unmodified DI17E6, and whereinthe CDR regions and heavy and light chain variable regions are at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% identical in their amino acid sequence compared to the respectivevariable regions of DI17E6. In addition, the invention also includesmodifications and variants of the DI17E6 antibody that are functionallyand/or pharmaceutically identical or similar to unmodified DI17E6, andwherein the constant regions are at least 80%, or at least 85%, or atleast 90%, or at least 95%, or at least 98%, or at least 99%, identicalin their amino acid sequence compared to the respective constant regionsof DI17E6. Changes in the constant regions of the IgG chains of theantibody may improve specific properties like immunogenicity, ADCC, andso on.

Preferably, the invention as described includes also modifications andvariants of the DI17E6 antibody that are functionally and/orpharmaceutically identical or similar to (unmodified) DI17E6 orabituzumab, and wherein the heavy and light chain variable regions areat least 95%, at least 98%, at least 99% or at least 99.5% identical intheir amino acid sequence compared to the respective heavy and lightchain variable regions of DI17E6. In addition, the invention alsoincludes modifications and variants of the DI17E6 antibody that arefunctionally and/or pharmaceutically identical or similar to unmodifiedDI17E6, preferably as described above in this paragraph, wherein theconstant regions are at least 90%, or at least 95%, or at least 99%, orat least 99.5%, or at least 99.9%, identical in their amino acidsequence compared to the respective constant regions of DI17E6. Changesin the constant regions of the IgG chains of the antibody may improvespecific properties like immunogenicity, ADCC, and so on.

Even more preferably, the invention as described includes alsomodifications and variants of the DI17E6 antibody that are functionallyand/or pharmaceutically identical or similar to (unmodified) DI17E6 orabituzumab, and wherein the CDR regions on the variable heavy and/orlight chain are at least 90%, at least 92%, at least 94%, at least 96%or at least 98% identical in their amino acid sequence compared to therespective CDR regions on the variable heavy and/or light chain regionsof DI17E6. In addition, the invention also includes modifications andvariants of the DI17E6 antibody that are functionally and/orpharmaceutically identical or similar to unmodified DI17E6, preferablyas described above in this paragraph, wherein the constant regions areat least 90%, or at least 95%, or at least 99%, or at least 99.5%, or atleast 99.9%, identical in their amino acid sequence compared to therespective constant regions of DI17E6.

Especially preferably, the invention as described includes alsomodifications and variants of the DI17E6 antibody that are functionallyand/or pharmaceutically identical or similar to (unmodified) DI17E6 orabituzumab, wherein the heavy and light chain CDR regions are 100%identical to (unmodified) DI17E6 or abituzumab, but wherein the heavyand light chain variable regions other than said CDR regions are atleast 95%, at least 98%, at least 99% or at least 99.5% identical intheir amino acid sequence compared to the respective heavy and lightchain variable regions of DI17E6. In addition, the invention preferablyalso includes modifications and variants of the DI17E6 antibody that arefunctionally and/or pharmaceutically identical or similar to unmodifiedDI17E6, preferably as described above in this paragraph, wherein theconstant regions are at least 90%, or at least 95%, or at least 99%, orat least 99.5%, or at least 99.9%, identical in their amino acidsequence compared to the respective constant regions of DI17E6.

Especially preferably, the invention as described includes alsomodifications and variants of the DI17E6 antibody that are functionallyand/or pharmaceutically identical or similar to (unmodified) DI17E6 orabituzumab, wherein the CDR regions on the variable heavy and/or lightchain are at least 90%, at least 92%, at least 94%, at least 96% or atleast 98% identical in their amino acid sequence compared to therespective CDR regions on the variable heavy and/or light chain regionsof DI17E6, and wherein the heavy and light chain variable regions otherthan said CDR regions are at least 95%, at least 98%, at least 99% or atleast 99.5% identical in their amino acid sequence compared to therespective heavy and light chain variable regions of DI17E6. Inaddition, the invention preferably also includes modifications andvariants of the DI17E6 antibody that are functionally and/orpharmaceutically identical or similar to unmodified DI17E6, preferablyas described above in this paragraph, wherein the constant regions areat least 90%, or at least 95%, or at least 99%, or at least 99.5%, or atleast 99.9%, identical in their amino acid sequence compared to therespective constant regions of DI17E6.

Especially preferably, the DI17E6 antibody or abituzumab is arecombinant, de-immunized monoclonal antibody of the IgG2 subclass asdescribed above and below which targets and inhibits ligand binding tohuman αv-integrins. Especially preferably, the carbohydrate structuresnormally present in the Fc region of said DI17E6 antibody or abituzumabhave been removed by genetically altering the amino acid residue thatnormally serves as the point of attachment rendering the moleculeaglycosylated. The antibody is especially preferably composed of 4polypeptide chains, 2 identical heavy chains consisting of 447 aminoacids each and 2 identical light chains consisting of 214 amino acidseach. Typically, the 4 chains are held together by a combination ofcovalent (disulfide) and non-covalent bonds. The approximate molecularweight of the molecule is 145 kDa.

More specifically, the DI17E6 antibody as described herein andespecially abituzumab or EMD 525797 is characterized as a high affinitypan-αv-integrin inhibitor, which has been shown in vitro to inhibitintegrin-dependent activation of latent TGF-β, to inhibit FMT, toprevent upregulation of integrins on myofibroblasts, and to blockcontraction of myofibroblasts. Thus, it specifically binds to a uniqueepitope that is specific for the human αv-integrin chain. Furthermore,it does not crossreact with other integrins such as the α4β1 and theplatelet fibrinogen receptor αIIbβ3 and preferably does not trigger ADCCand CDC.

According to the invention, DI17E6 is believed to be highly effective inpatients suffering from fibrotic diseases, more preferably systemicsclerosis and especially in one or more of the indications of systemicsclerosis described herein. More specifically, abituzumabis believed tobe highly effective in patients suffering from fibrotic diseases, morepreferably systemic sclerosis and especially in one or more of theindications of systemic sclerosis described herein.

Furthermore, it is believed that DI17E6 as described herein, andespecially abituzumab, is suitable to provide a more effective and/orsafer therapy od fibrotic diseases as described herein, will be bettertolerated and can provoke better immune response than seen with thetherapies previously known. Preferably, treatment with abituzumab isbelieved to be more effective, safer, will be better tolerated and canprovoke better immune responses in the treatment of patients withSSc-ILD, preferably also patients who already receive constant doses ofmycophenolate.

As described in the instant invention, TGF-β is shown to be the mastermediator of tissue fibrosis. Together with the findings that in patientswith SSc, the TGF-β signalling pathways are activated, and that some ofthe αvβx integrins heterodimers control the activation of TGF-β, theanti-αv integrin antibody DI17E6, or a biologically active variant ormodification thereof, is believed to have a beneficial effect in SSc dueto its shown interference with TGF-β activation, and additionally dueits TGF-independent functions on αv integrins that are found to beinvolved in fibrosis. Therefore, EMD 525797 may be beneficial inpatients with SSc through multiple modes of action. This is described inmore detail above and/or below. However, DI17E6 cannot be directlytested in rodent models of fibrosis because of lacking cross-reactivity(see section A3.1.3 Mode of action). Preclinical models of fibrosis innonhuman primates for SSc or SSc-ILD are deemed difficult, if notunfeasible. Preclinical experiments with blocking αv antibodies otherthan DI17E6 have been performed with human cells but contradictingresults from in vivo experiments limit their relevance. Thus, herein wedescribe i.a. the effect of genetic or surrogate antibody mediated αvintegrin blockade in preclinical fibrosis models in rodents, and,additionally, that there is more evidence for αv integrin blockade tosupport the SSc-ILD disease indication (acute lung injury responses,aberrant epithelial wound closure response, EMT) than SSc manifestationsin other organs.

Although antibody mediated blockade of αvβ3 and αvβ5 integrins in vitrowas shown to inhibit TGF-β dependent responses in human fibroblaststaken from tissue samples of either SSc or idiopathic pulmonary fibrosis(IPF) patients (Asano et al., American Journal of Pathology, Vol. 168,No. 2, February 2006, The Journal of Immunology, 2005, 175: 7708-7718,ARTHRITIS & RHEUMATISM, Vol. 52, No. 9, September 2005, pp 2897-2905,Journal of Investigative Dermatology (2006) 126, 1761-1769, Scotton etal., J. Clin. Invest. 119:2550-2563 (2009), so far, there is noconvincing evidence that these integrins activate TGF-β in vivo. Forexample, the genetic deletion of αvβ3 and αvβ5 does not have an effectin bleomycin-induced lung fibrosis (Atabai et al., J. Clin. Invest.119:3713-3722 (2009). However, the combination of genetic ablation of β8integrin (Itgb8) with antibody inhibition of αvβ6 was shown to result ina more severe phenotype than in Itgb8−/− animals but a similar phenotypeas seen in Tgfb1 null mice (Aluwihare et al., Journal of Cell Science122, 2009, 227-232), discussing whether either or both integrins mayplay a role in the activation of TGF-β in the biological development ofin mice in the different states of differentiation from embryo to adultmice in vivo.

Takahashi et al. (Am. J. Respir. Cell Mol. Biol. Vol. 24, pp. 264-271,2001) investigated the role of osteopontin in the pathogenesis ofbleomycin-induced pulmonary fibrosis in mice. Osteopontin (OPN) isreported to be one of the cytokines produced by activated macrophagesand mediates various functions, including cell attachment and migration,by interacting with av integrin.

These effects of OPN on fibroblasts were significantly suppressed byaddition of antimouse av integrin monoclonal antibody (RMV-7) in thismodel.

Transforming growth factor beta (TGF-β) is an important driver ofpulmonary fibrosis and therapeutic strategies to inhibit its actions aresought. However, TGF-β has other homeostatic roles that could maketherapeutic inhibition problematic. Horan et al. (Am J Respir Crit CareMed Vol 177. pp 56-65, 2008) thus investigated Inhibition of theintegrin avb6, a key activator of TGF-β in lung, using a monoclonalpSmad2/3 primary antibody that blocks avb6-mediated TGF-β activation, inmurine bleomycin-induced lung fibrosis model. The partial inhibition ofTGF-β using said avb6 integrin antibody was described as effective inblocking murine pulmonary fibrosis without exacerbating inflammation insaid murine model.

Synthetic peptides containing the RGD sequence described to be able toinhibit integrin-related function in different cell systems. Moon et al.(Respiratory Research 2009, 10:18) investigated the effects of syntheticArg-Gly-Asp-Ser (RGDS) peptide on key inflammatory responses tointratracheal (i.t.) lipopolysaccharide (LPS) treatment and on theintegrin signaled mitogen-activated protein (MAP) kinase pathway duringthe development of acute lung injury in mice. A pretreatment with RGDSinhibited LPS-induced increases in neutrophil and macrophage numbers,total protein levels and TNF-α and MIP-2 levels, and matrixmetalloproteinase-9 activity in bronchoalveolar lavage (BAL) fluid at 4or 24 h post-LPS treatment. Importantly, the inhibition of theinflammatory responses and the kinase pathways were still evident whenthis peptide was administered 2 h after LPS treatment. Similarly, ablocking antibody against αv integrin significantly inhibitedLPS-induced inflammatory cell migration into the lung, proteinaccumulation and proinflammatory mediator production in BAL fluid, at 4or 24 h post-LPS. These results suggest that RGDS with high specificityfor αv integrins attenuates inflammatory cascade during LPS-induceddevelopment of acute lung injury in this murine model.

Activation of latent TGF-β by the αvβ6 integrin has been reported to beone critical step in the development of acute lung injury. Jenkins etal. (J. Clin. Invest. 116:1606-1614 (2006) show that thrombin, and otheragonists of protease-activated receptor 1 (PAR1), activate TGF-β in anαvβ6 integrin-specific manner. This effect is PAR1 specific and isbelieved to be mediated by RhoA and Rho kinase. Intratrachealinstillation of the PAR1-specific peptide TFLLRN increases lung edemaduring high-tidal-volume ventilation, and this effect is described to becompletely inhibited by a blocking antibody against the αvβ6 integrin.However, activation of TGF-β by αvβ6 probably requires more than simplybinding to LAP, as we have identified cytoplasmic mutants that bind LAPbut do not activate TGF-β. Furthermore, the αvβ1 and α8β1 integrins areboth able to bind LAP but do not activate TGF-β. The PAR1-mediatedenhancement of αvβ6-dependent TGF-β activation found in these in vitroexperiments might be interpreted as one mechanism by which activation ofthe coagulation cascade contributes to the development of acute lunginjury.

Transforming growth factor (TGF)-β family members regulate multipleaspects of wound repair through effects on cell proliferation, matrixproduction, and tissue inflammation, but the effects of TGF-β on woundclosure itself have been controversial. Neuohr et al (Am. J. Respir.Cell. Mol. Biol. Vol. 35, pp 252-259, 2006) report that the usedblocking antibodies to TGF-13 enhanced the degree of closure of scratchwounds in primary airway epithelial monolayers in this airway epithelialcell culture model, while addition of exogenous TGF-1β inhibited thedegree of closure, suggesting that endogenous activation of TGF-β mightserve as a brake on the degree of wound closure. Blockade of TGF-β1enhanced the degree of wound closure, whereas blockade of TGF-β2 had noeffect in this cell culture model. Here, TGF-β1 (but not TGF-β2) couldbe activated by two members of the integrin family, αvβ6 and αvβ8, whichboth were found to be expressed on said airway epithelial cells. In thedescribed model, wounding of the cell layer induced activation of TGF-βthrough effects of both integrins, but the chosen mouse monoclonalantibody against human αvβ8 (37E1) enhanced the degree of wound closure,whereas the chosen mouse monoclonal antibodies against human αvβ6(anti-HEL-AB MSB0011523H-1, 10D5) did not.

Transforming growth factor-b1 (TGF-β1) is a potent mediator of thedifferentiation of fibroblasts into myofibroblasts, which ischaracterized by the appearance of the cytoskeletal protein α-smoothmuscle actin. Lygoe et al. (Wound Rep. Reg. 2004; 12:461-470) describethat blockade of the αv and/or 131 integrins with selected monoclonalmouse anti-human antibodies (LM609 against αvβ3, P4C10 against β1, P1F6against αvβ5, A11B2 against β1, L230 against αv) prevented theTGF-β1-induced myofibroblast differentiation, seen by the increasedexpression of α-smooth muscle actin and enhanced collagen gelcontraction in three human fibroblast cell lines (from the mouth, skin,and kidney). Further, blockade of αv specific integrins αvβ5 and αvβ3suppressed myofibroblast differentiation in fibroblasts from the mouthand skin; however, in the kidney cells, the prevention ofdifferentiation was seen only with blockade of αvβ5 integrin but notαvβ3. These data indicate a role for αv integrins in the differentiationof human fibroblasts from the mouth, skin, and kidney intomyofibroblasts and suggest that there might be a common differentiationpathway.

αv integrins might also participate in the optimal function of theangiogenesis through interaction with various growth factors such asPlatelet Derived Growth Factor (PDGF), Vascular Endothelial GrowthFactor (VEGF) and Fibroblast Growth Factor (FGF) factor. As BIBF-1120, anew chemical entity (NCE) from Boehringer Ingelheim, targets thetyrosine kinase of these receptors and has shown preliminary efficacy ina PoC trial in idiopathic pulmonary fibrosis (IPF), another form of lungfibrosis with features in common with SSc-ILD, DI17E6 is expected tohave a potential beneficial effect through inhibition of thesereceptors. This is in line with inhibition of VEGF-induced ERKphosphorylation by DI17E6 observed with human cells.

Without being bound by the below discussed mechanism, based on theresults underlying the instant invention, we believe thatEpithelial-to-mesenchymal transition (EMT) contributes to fibrosis,fibrotic disorders and/or fibrosing lung disorders. TGF-β and αvβxintegrins are believed to be involved in this transdifferentiationprocess and that blocking of the function of these integrins by theanti-αv integrin antibody DI17E6, or a biologically active variant ormodification thereof downregulates EMT and thus become beneficial infibrosis, fibrotic disorders and/or fibrosing lung disorders.

Oxidative stress is believed to play a role in fibrotic diseases. Cellsexpressing αvβx integrins are believed to be protected from oxidativestress induced apoptosis. It appears therefore to be possible thatagents blocking the function of these integrins might increase apoptosisof fibroblasts and myofibroblasts.

Some of the αvβx integrins are found to be overexpressed in SSc-ILD andother forms of lung fibrosis, and the respiratory epithelium in lungbiopsies from patients with SSc-ILD and IPF expresses increased levelsof αvβ6 by immunohistochemistry. Increased numbers of αvβ3, αvβ5 and β6expressing T cells are found in the bronchoalveolar lavage fluid frompatients with SSc compared to normal controls (Luzina et al., Am JRespir Crit Care Med Vol 177. pp 56-65, 2008. However, it has been shownthat β6 integrin knockout mice develop lung inflammation, but do notproceed to develop pulmonary fibrosis, after bleomycin exposure (Luzinaet al., ARTHRITIS & RHEUMATISM, Vol. 48, No. 8, August 2003, pp2262-2274).

Dermal fibroblasts from patients with SSc display enhanced expression ofαvβ3 and αvβ5, and elevated TGF-β activation, which is inhibited byblocking antibody P1F6 directed against αvβ5 (Asano et al., ARTHRITIS &RHEUMATISM Vol. 52, No. 9, September 2005, pp 2897-2905; Journal ofImmunology, 2005, 175: 7708-7718; American Journal of Pathology, Vol.168, No. 2, February 2006; Journal of Investigative Dermatology (2006)126, 1761-1769). Increased immunostaining with an anti-αvβ5 antibody isfound in fibroblastic foci in lungs of patients with IPF (Scotton etal., J. Clin. Invest. 119:2550-2563 (2009)).

Cartilage oligomeric protein (COMP) is an extracellular matrix (ECM)protein that resides in cartilage, tendon, and other connective tissue.COMP is overexpressed in skin biopsies from patients with SSc. In SSc,serum COMP levels are elevated, predict mortality, and correlate withlung function decline and skin fibrosis as measured with the modifiedRodnan Skin Score (mRSS). COMP is also one of the four signature RNAs inSSc skin that predict severity of skin involvement.

Yang et al. describe that Periostin facilitates skin sclerosis viaPI3K/Akt dependent mechanism in a mouse model of scleroderma (Yang. L,Serada S, Fujimoto M, Terao M, Kotobuki Y, et al. (2012) PLoS ONE 7(7):e41994 doi:10.1371/journal.pone.0041994). Using skin from patients andhealthy donors, also the expression of periostin was assessed byimmunohistochemistry and immunoblotting analyses. Furthermore, it wasfound that recombinant mouse periostin directly induced Col1a1expression in vitro, and this effect was inhibited by blocking the αvintegrin-mediated PI3K/Akt signaling either with anti-αv functionalblocking antibody or with the PI3K/Akt kinase inhibitor LY294002. As aresult, it is concluded that Periostin plays an essential role in thepathogenesis of Bleomycin-induced scleroderma in mice and that Periostinmay represent a potential therapeutic target for human scleroderma.

Wu et al. (Journal of Investigative Dermatology (2012) 132, 1605-1614)investigated the role of osteopontin (OPN), a matricellular protein withproinflammatory and profibrotic properties, in systemic sclerosis anddermal fibrosis in mice in the bleomycin-induced fibrosis model.According to that, OPN-deficient mice developed less dermal fibrosiscompared with wild-type (WT) mice in said fibrosis model. Finally, theyfound TGF-β production by OPN-deficient macrophages to be reducedcompared with WT. In conclusion, OPN levels are reported to be increasedin SSc patients, and the data obtained are deemed to suggest that OPNmight play an important role in the development of dermal fibrosis inmice, and that OPN thus might be a therapeutic target in SSc.

Plasma of a total of 70 patients with SSc was analysed by Lorenzen etal. (Rheumatology 2010; 49:1989-1991 doi:10.1093/rheumatology/keq223Advance Access publication 20 Jul. 2010), with twenty age-matchedhealthy volunteers and 59 patients with idiopathic pulmonaryhypertension as controls. The results may indicate the possibility ofusing OPN inhibitors as a novel therapeutic target of T-cell chemotaxis,since monoclonal OPN antibodies have been used in collagen-inducedarthritis models. In conclusion, OPN levels were described to parallelthe development of pulmonary fibrosis in SSc and thus might be anattractive biomarker of this complication.

In summary, based on the results and data underlying the instantinvention, we strongly believe that the specific interaction of DI17E6,and preferably also the biologically active variants or modificationsthereof as described herein, with the αvβx integrins controls theactivation of TGF-β and multiple additional mechanisms in a specificallyadvantageous manner that addresses the pathogenesis of fibrosis,fibrotic disorders, including SSc. Therefore, an advantageouslybeneficial effect of said interaction with the signalling in fibrosis,fibrotic disorders, preferably SSc, and in particular in SSc-ILD, isexpected.

The excellent safety and tolerability of EMD 525797 in various cancerindications is expected to be similar in fibrotic diseases, includingSSc and especially including SSc-ILD. Together with the expected benefitof EMD 52579, the benefit/risk ratio of EMD 525797 in SSc-ILD should bepositive in SSc-ILD and greater than for CYC. EMD 525797 is thusbelieved to be also beneficial on other manifestations of SSc.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; mouse gonadotropin-associatedpeptide; inhibin; activin; vascular endothelial growth factor (VEGF);integrin; thrombopoietin (TPO); nerve growth factors such as NGFβ;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; erythropoietin (EPO); interferons such as IFNα, IFNβ, andIFNγ; colony stimulating factors such as M-CSF, GM-CSF and G-CSF;interleukins such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-12; and TNFα or TNFβ. Preferred cytokinesaccording to the invention are interferons and TNFα.

An “anti-angiogenic agent” refers to a natural or synthetic compoundwhich blocks, or interferes with to some degree, the development ofblood vessels. The anti-angiogenic molecule may, for instance, be abiological molecule that binds to and blocks an angiogenic growth factoror growth factor receptor. The preferred anti-angiogenic molecule hereinbinds to a receptor, preferably to an integrin receptor or to VEGFreceptor. The term includes according to the invention also integrin(receptor) inhibitors.

The term “integrin inhibitors” or “integrin receptor inhibitors” refersto a natural or synthetic molecule that blocks and inhibit an integrinreceptor. In some cases, the term includes antagonists directed to theligands of said integrin receptors (such as for α_(v)β₃: vitronectin,fibrin, fibrinogen, von Willebrand's factor, thrombospondin, laminin;for α_(v)β₅: vitronectin; for α_(v)β₁: fibronectin and vitronectin; forα_(v)β₆: fibronectin). Antagonists directed to the integrin receptorsare preferred according to the invention. Integrin (receptor)antagonists may be natural or synthetic peptides, non-peptides,peptidomimetica, immunoglobulins, such as antibodies or functionalfragments thereof, or immunoconjugates (fusion proteins). Preferredintegrin inhibitors of the invention are directed to receptor of α_(v)integrins (e.g. α_(v)β₃, α_(v)β₅, α_(v)β₆ and sub-classes). Preferredintegrin inhibitors are α_(v) antagonists, and in particular α_(v)β₃antagonists. Preferred α_(v) antagonists according to the invention areRGD peptides, peptidomimetic (non-peptide) antagonists and anti-integrinreceptor antibodies such as antibodies blocking α_(v) receptors.Exemplary, non-immunological α_(v)β₃ antagonists are described in theteachings of U.S. Pat. Nos. 5,753,230 and 5,766,591. Preferredantagonists are linear and cyclic RGD-containing peptides. Cyclicpeptides are, as a rule, more stable and elicit an enhanced serumhalf-life. A preferred further integrin antagonist of the invention is,however, cyclo-(Arg-Gly-Asp-DPhe-NMeVal) (EMD 121974, Cilengitide®,Merck KGaA, Germany; EP 0770 622) which is efficacious in blocking theintegrin receptors α_(v)β₃ and α_(v)β₅, and to a lesser extend α_(v)β₆,α_(v)β₈, α_(v)β₃. A combination therapy of DI17E6 together withCilengitide in fibrotic disease patients can be effective according tothe invention.

DI17E6 is administered usually by intravenous injection, however otheradministration forms convenient in the art for antibody/protein drugsare applicable. All standard infusion solutions and formulation areapplicable, such as described in WO 2005/077414 or WO 2003/053465,including liposomal formulations. It is, in addition, favorable toprovide human serum albumin nanoparticles loaded with DI17E6 andoptionally (to increase cytotoxicity) chemotherapeutic drugs(Biomaterials 2010, 8, 2388-98; Wagner et al.).

Especially preferred as the anti-αv integrin antibody DI17E6, or DI17E6,according to the instant invention is the antibody with the registeredInternational Non-Proprietary Name (INN) Abituzumab.

In connection with the instant invention, Abituzumab is a recombinant,de-immunized monoclonal antibody of the IgG2 subclass that targets andblocks ligand binding to the human αv-integrins. In addition, thecarbohydrate structures normally present in the Fc region have beenremoved by genetically altering the amino acid residue that normallyserves as the point of attachment rendering the molecule aglycosylated.Accordingly, the antibody Abituzumab is preferably composed of fourpolypeptide chains, two preferably identical heavy chains consisting of447 amino acids each and two preferably identical light chainsconsisting of 214 amino acids each. The four chains are held together bya combination of covalent (disulfide) and non-covalent bonds. Theapproximate molecular weight of the molecule is 145 kDa.

The antibody Abituzumab can be produced by mammalian cell culture in aserum-free growth medium. The antibody be purified by affinity andion-exchange chromatography. The process may also include specific viralinactivation and removal steps. The antibody Abituzumab may be thentransferred into formulation buffer and brought to the desiredconcentration.

Description and Composition of a Preferred Medicinal Product:

Abituzumab, e.g. in the concentration of 250 mg/10 mL can be used as thefinal drug product as a sterile solution intended for intravenous (i.v.)administration. Abituzumab drug product can be supplied in a 30 R type Iglass vial, e.g. closed with a grey butyl rubber stopper and for examplesealed with an aluminum/red polypropylene flip-off seal. Such vials canbe used as single-use vial contains, e.g. containing 250 mg ofAbituzumab as a 25 mg/mL preservative-free citrate buffered salinesolution, for example containing polysorbate 80 (Tween® 80) asstabilizer. Such vials may contain a sufficient overage to remove a 10mL volume of Abituzumab final drug product. Generally, for such a finaldrug product of Abituzumab, only excipients are used that are regardedas safe and conform to current European Pharmacopeia (EP) or UnitedStates Pharmacopeia (USP) or both.

Especially preferably, Abituzumab is a humanized IgG2 antibody that isgenetically modified not to induce antibody-dependent cell cytotoxicity(ADCC) or complement-dependent cytotoxicity (CDC). Said Abituzumabantibody binds to the human αv-integrin receptor subunit with highspecificity, thereby inhibiting ligand binding to the αv heterodimers(αvβ1, αvβ3, αvβ5, αvβ6, αvβ8). It specifically inhibits αv-integrinsand blocks αv-integrin-mediated cell attachment and migration. It doesnot cross-react with other integrins, including the platelet fibrinogenreceptor αIIbβ3, and recognizes only human and monkey αv-integrins.Abituzumab recognizes an epitope on the αv-integrin receptor subunitthat is not located in or close to the ligand pocket. Therefore, itsmechanism of action differs from that described for pureligand-competing antagonists such as cyclic RGD peptides. The integrinsαvβ3 and αvβ5 are selectively expressed on activated endothelial cells(EC), on resting platelets, smooth muscle cells, in the thyroid, on somekidney endothelia and epithelia, on the fallopian tube endothelium, andon osteoclasts. Furthermore, αv-integrins are expressed to a variableextent on malignant cells from different tumor entities, including thosefrom colorectal cancer and prostate cancer. Accordingly, the binding ofAbituzumab to its target preferably functionally blocks the integrinreceptor and thus inhibits its binding to the correspondingextracellular matrix (ECM) ligand (i.e. vitronectin).

Immunohistochemical (IHC) examinations in humans and in different animalspecies showed that Abituzumab is highly species-specific: Abituzumabbinds only to human and cynomolgus monkey αv-integrins, with comparablecross-reactivity between human and monkey tissues. Abituzumab has beenshown in vitro to interfere with several aspects involved in tumorangiogenesis, such as EC attachment to ECM, destabilization of focalcontacts, EC migration, transmission of angiogenic growth factorsignaling (VEGF-induced ERK phosphorylation) and EC viability.Abituzumab also directly affects the tumor cells. In vitro experimentsrevealed that Abituzumab can affect cell adhesion to the ligands ofαv-integrins and proliferation of tumor cells.

In vivo, the anti-tumor effect of Abituzumab was evaluated usingdifferent αv-integrin expressing tumor cell lines for tumor xenograftmodels (e.g., melanoma, NSCLC, CRC, prostate cancer). As Abituzumab isspecific for human and monkey αv-integrins, its anti-angiogenic activitycannot be studied in conventional rodent xenograft tumor models.Therefore, the xenograft tumor experiments in mice demonstrated solelythe potential anti-tumor activity of Abituzumab. Abituzumab was able toinhibit the growth in in vivo tumor experiments using cancer cell linesor primary explants of different indications (e.g., melanoma, NSCLC,prostate cancer and CRC).

The anti-angiogenic mechanism of action of Abituzumab was evaluated in ahuman skin xenograft/tumor cell line experiment in the absence of thetarget αv-integrins on the malignant cells. Abituzumab inhibited thegrowth of human αv-integrin-deficient melanoma cells injected into humanskin that had been transplanted onto SCID mice. Because of the absenceof the target on the tumor cells themselves, Abituzumab can target onlythe human endothelial cells, and tumor growth reduction is most likelycaused by inhibition of tumor angiogenesis. Systemic administration ofAbituzumab in cynomolgus monkeys blocked the induction of angiogenesisin subcutaneously implanted Matrigel plugs containing angiogenic growthfactors to stimulate angiogenesis.

Especially preferred according to the invention are subjects asdescribed herein, wherein the characteristics of two or more preferred,more preferred and/or especially preferred embodiments, aspects and/orsubjects are combined into one embodiment, aspect and/or subject.Preferably, according to this invention, preferred subjects orembodiments can be combined with other preferred subjects orembodiments; more preferred subjects or embodiments can be combined withother less preferred or even more preferred subjects or embodiments;especially preferred subjects or embodiments can be combined with otherjust preferred or just even more preferred subjects or embodiments, andthe like.

The term “about” as used herein with respect to numbers, figures, rangesand/or amounts is preferably meant to mean “circa” and/or“approximately”. The meaning of those terms is well known in the art andpreferably includes a variance, deviation and/or variability of therespective number, figure, range and/or amount of plus/minus 15% andespecially of plus/minus 10%.

In any case, the term “about” as used herein with respect to numbers,figures, ranges and/or amounts is preferably meant to mean “circa”and/or “approximately”. The meaning of those terms is well known in theart and preferably includes a variance, deviation and/or variability ofthe respective number, figure, range and/or amount of at leastplus/minus 5%.

The terms “disorder(s)” and “disease(s)” as used herein are well-knownand understood in the art. In the context of the present invention theyare preferably used as synonyms and thus are preferably interchangeable,if the context they are used herein does not strongly implicateotherwise. Accordingly, the terms “fibrotic disorder(s)” and “fibroticdisease(s)” as used herein are also well-known and understood in theart. In the context of the present invention they are preferably used assynonyms and thus are preferably interchangeable, if the context theyare used herein does not strongly implicate otherwise.

In the medical context, including, but not limited to treatmentregimens, dosing schedules and clinical trial designs, for convenienceand/or ease of use by patients, medical staff and/or physicians, as wellas reliability and/or reproducibility of results etc., the terms“week”/“a week”, “month”/“a month” and/or “year”/“a year” can used withslight deviations from the definitions of the Gregorian calendar. Forexample, in said medical context, a month is often referred to as 28days, and a year is often referred to 48 weeks.

Thus, in the context of the instant invention, the term “week” or “aweek” preferably refers to a period of time of about 5, about 6 or about7 days, more preferably about 7 days.

In the medical context, the term “month” or “a month” preferably refersto a period of time of about 28, about 29, about 30 or about 31 days,more preferably about 28, about 30 or about 31 days.

In the medical context, the term “year” or “a year” preferably refers toa period of time of about 12 months or to a period of time of about 48,about 50, or about 52 weeks, more preferably 12 months, or about 48 orabout 52 weeks.

The invention is explained in greater detail below by means of examples.The invention preferably can be carried out throughout the range claimedand is not restricted to the examples given here.

Moreover, the following examples are given in order to assist theskilled artisan to better understand the present invention by way ofexemplification. The examples are not intended to limit the scope ofprotection conferred by the claims. The features, properties andadvantages exemplified for the processes, compounds, compositions and/oruses defined in the examples may be assigned to other processes,compounds, compositions and/or uses not specifically described and/ordefined in the examples, but falling under the scope of what is definedin the claims.

Thus, the following examples describe the invention in more detail butdo not limit the invention and its scope as claimed.

EXAMPLES Example 1 Myofibroblast Differentiation is Induced inEpithelial-Fibroblast Co-Cultures and is Inhibited by Abituzumab List ofAbbreviations αSMA: Alpha Smooth Muscle Actin BSA: Bovine Serum AlbuminCy5: Cyanine 5

DAPI: 4′,6-diamidino-2-Phenylindole

ECM: Extracellular Matrix FGM: Fibroblast Growth Medium FMT: Fibroblastto Myofibroblast Transition FITC: Fluorescence Isothiocyanate IXM: ImageXpress Micro Screening System ug: Micgrogram mg: Milligram mL:Milliliter ng: Nanogram NHDF: Normal Human Dermal Fibroblasts NHLF:Normal Human Lung Fibroblasts PBS: Phosphate-Buffered Saline TGF-β1:Transforming Growth Factor-Beta 1 ELISA: Enzyme-Linked ImmunosorbentAssay FBS: Fetal Bovine Serum IL Interleukin Summary

TGF-β1 is a potent mediator of fibroblast to myofibroblast transition(FMT) which contributes to increased extracellular matrix deposition andis main driver of fibrotic diseases. There is substantial evidence forcrosstalk between αv integrins and TGF-β during these processes. TGF-βis secreted in a latent form which contains a Latency Associated Peptide(LAP) region. The LAP of TGF-β1 contains an RGD motif which interactswith the integrins αvβ1, αvβ3, αvβ5, αvβ6 and αvβ8 resulting inactivation of TGF-β1. Abituzumab is a human antibody specific for αv andtherefore inhibits αvβ1, αvβ3, αvβ5, αvβ6 and αvβ8.

The ability of abituzumab to block FMT was examined using an epithelialcell/fibroblast co-culture, mimicking the potential interaction ofepithelial cells and fibroblasts in tissues undergoing fibrosis.Co-culture of NCI-H358 or Calu3 cells with fibroblasts resulted ininduction of aSMA and multiple mRNA transcripts that are markers for FMTand also increased IL-6 production. In this system these markers werereduced by abituzumab treatment, demonstrating that αv integrins play arole in FMT.

Introduction

Fibrotic diseases are characterized by excessive scarring due toproduction, deposition and contraction of extracellular matrix and isbelieved to be driven by myofibroblast proliferation and activation.Fibrotic diseases represent one of the largest groups of diseases forwhich there is no effective therapy. The fibrotic processes is regulatedby complex set of interactions within a network of profibrotic andantifibrotic mediators. TGF-β signaling is believed to play an importantrole in fibroblast to myofibroblast transition (FMT) which contributesto increased extracellular matrix deposition and is main driver ofdisease.

TGF-β isoforms are synthesized as latent precursors complexed withlatent TGF-β binding proteins, which contains a Latency AssociatedPeptide (LAP) region. There is substantial evidence for crosstalkbetween αv integrins and TGF-β during these processes. The LAP of TGF-β1contains an RGD motif which interacts with the integrins αvβ1, αvβ3,αvβ5, αvβ6 and αvβ8 resulting in activation of TGF-β1. Abituzumab is apan-αv integrin antibody that binds allosterically to the ligand-bindingαv subunit and thus prevents ligand from binding to all αvβ heterodimersand therefore inhibits αv integrin-dependent activation of latent TGF-βand thus blocks acquisition of the myofibroblast phenotype byfibroblasts and other precursors.

Nature and Purpose of Study

The purpose of the present study was to determine the ability ofabituzumab to block TGF-β activation and FMT in vitro and thus showingits potential as a therapeutic agent for fibrotic diseases.

Materials and Methods Test Systems

The test system is epithelial cell/fibroblast co-culture, mimicking thepotential interaction of epithelial cells and fibroblasts in tissuesundergoing fibrosis. Normal human lung fibroblast (NHLF) or Normal humandermal fibroblast (NHDF) from healthy donors were cocultured withNCI-H358 or Calu 3 cell line.

Tissue Description Company Cat # Lot# # Normal human Lonza/cloneticsCC-2512 0000374386 26789 lung fibroblasts Normal human Lonza/cloneticsCC-2512 0000343490 25745 lung fibroblasts Normal human Lonza/cloneticsCC-2512 0000369145 26646 lung fibroblasts Normal human Lonza/cloneticsCC-2511 0000399829 27240 dermal fibroblasts Normal human Lonza/cloneticsCC-2511 0000352805 26086 dermal fibroblasts Normal human Lonza/cloneticsCC-2511 0000281135 23920 dermal fibroblasts NCI-H358 Merck KGaA, ATCC-NCI-H358 N/A bronchioalveolar Cell Culture CRL- P4 WCB cell line; FromStrain Collection, 5807 08.07.2013 human, non-small So-ABB4 cell lungcarcinoma Calu-3 airway Merck KGaA, ATCC Calu-3/10 N/A epithelial cellline; Cell Culture HTB 55 02.04.08 From human lung Strain Collection,adenocarcinoma So-ABB4

Test Material and Stimulus

Lot/ Antibodies Company Accession# Abituzumab (DI17E6) EMD Serono 402499Anti-HEL IgG2 EMD Serono A12-145-2

Supplies and Instruments

Material/Reagent Provider Cat. # Lot # FGM Fibroblast Growth Lonza/CC-3132    354178 Medium and Bullet Kit clonetics (include Cat#CC-3131 &CC-4126) Fibroblast Growth Medium Lonza/ CC-3131    409065 cloneticsFibroblast Bullet Kit Lonza/ CC-4126    417247 cloneticsGentamicin-Sulfate Lonza/ CC-4081J    417245 clonetics Insulin Lonza/CC-4021J    417243 clonetics rhFGF Lonza/ CC-4065J    417244 cloneticsFBS Lonza/ CC-4101J    417246 clonetics RPMI medium 1640 Life 11875-093  1627680 Technology Heat Inactivated Fetal Corning 35-076-CV  35076102Bovine Serum Sodium Pyruvate Life 11360-070   1585688 Technology DMEMLife 11965-092   1621394 Technology Reagent Pack/Subculture Lonza/CC-5034    409088 Reagents clonetics Hepes Buffered Saline Lonza/CC-5022    362157 clonetics Trypsin Lonza/ CC-5012    372708 cloneticsTrypsin Neutralizing Lonza/ CC-5002    385562 Solution clonetics T 150cm2 flasks Corning 430825  19514018 UltraPure Distilled Water Invitrogen10977-015   1607416 PBS Invitrogen 20012-027   1457916 Collagen 1 Cellware BD 356700   2136233 96-well Black/Clear Plate Biosciences 24 wellplates Falcon 353047 N/A Odyssey Blocking Buffer Li-COR 927-40000 T1752(500 mL) BD Cytofix/Cytoperm BD 554714   3099684Fixation/Permeabilization Biosciences kit Fixation Solution BD 554722  3231840 Biosciences Permeabilization (Perm) BD 554723   4174605 WashBuffer Biosciences Anti-actin, smooth muscle, Millipore CBL171   2377949clone ASM-1 Goat Anti-mouse IgG Alexa Invitrogen A11001   1503602Fluor488: Invitrogen Corp Goat Anti-rabbit IgG Alexa Invitrogen A21245  1623067 Fluor647: Invitrogen Corp DAPI, dilactate Sigma D9564-042M4005 Aldrich 10MG RNeasy 96 kit Qiagen 74181 148019008 RT2 FirstStrand kit Qiagen 330401 N/A RT2 Profiler PCR Array Qiagen 12473 N/A(Custom) Equipment: Company Serial # Allegra X-14R Centrifuge BeckmanCoulter B34604-AA Laminar hood model 1395, Thermo Fisher 114627-128 1300SeriesA2 Incubator at 37 C. with Thermo Fisher 313910-5441 humidified 5%CO2 Spectramax M5e Molecular MVE06101 Devices Biotek 405LS microplatePerkin Elmer 1306126 washer Microscope, Nikon TMS DSC Optical 11345Services Water Bath Equipnet 35933-2-2 Image Xpress Micro Molecular12264 Imaging Microscope Devices Optiplex 990 Image Dell X16-96076Analysis Computer QuantStudio 12K Flex Applied 285880466 Biosystems byLife Technologies

Experimental Design Outline Study Design

Fibroblast culture media (FGM with 2% FBS): FGM™-2 BulletKit™ (CC-3132)contains one 500 ml bottle of Fibroblast Cell Basal Medium (CC-3131) andFibroblast Bullet Kit (CC-4126) with the following growth supplements:0.5 ml hFGF-B (CC-4065J); 0.5 ml Insulin (CC-4021J); 10 ml FBS(CC-4101J); 0.5 ml GA-1000 (CC-4081J).

Fibroblast culture media for FMT assays (FGM with 0.1% FBS): FGM™-2BulletKit™ (CC-3132) contains one 500 ml bottle of Fibroblast Cell BasalMedium (CC-3131) and Fibroblast Bullet kit (CC-4126) with the followinggrowth supplements: 0.5 ml hFGF-B (CC-4065J); 0.5 ml Insulin (CC-4021J);0.5 ml FBS (CC-4101J); 0.5 ml GA-1000 (CC-4081J).

NCI-H358 medium: 500 ml RPMI with 55 ml of heat inactivated FBS+5 ml ofsodium pyruvate

Calu-3 media: 500 ml DMEM with 55 ml of heat inactivated FBS

NHLF and NHDF were grown in T150 tissue culture flasks in FGM with 2%FBS until they were 70-80% confluent on the day of the assay. The cellswere rinsed with 6 ml HEPES buffered saline solution (LonzaCat#CC-5022), trypsinized with 6 ml trypsin/EDTA (Lonza Cat#CC-5012) for5 min at room temperature. The trypsin was inactivated with 6 ml trypsinneutralizing solution (Lonza Cat#CC-5002), spun down at 400 g for 4 minand washed once with FGM with 2% FBS.

For imaging studies, fibroblasts (NHLF or NHDF) were seeded at 10,000cells/well in Collagen 1 Cellware 96-well Black/Clear Plate. Cells werecultured for 8 hours in FGM with 2% FBS. Media were aspirated and cellswere starved overnight in FGM containing 0.1% FBS. Media were aspiratedand 100 ul of FGM with 0.1% FBS containing abituzumab or anti-HEL IgG ata conc of 20 ng/ml were added and incubated for 30 min. For coculture:NCI-H358 or Calu-3 were plated into the wells containing fibroblast at adensity of 2,000 cells in 100 ul of NCI-H358 or Calu-3 media. 5 dayslater, media were collected and stored at −80 C for IL-6 detection.Cells were fixed and stained according to the alpha smooth muscle actin(αSMA) staining procedure.

For gene expression studies, fibroblasts (NHLF or NHDF) were seeded at200,000 cells/well in 24 wells culture plates. Cells were cultured for 8hours in FGM with 2% FBS. Media were aspirated and cells were starvedovernight in FGM containing 0.1% FBS. Media were aspirated and 300 ul ofFGM with 0.1% FBS containing abituzumab or anti-HEL IgG at a conc of 20ng/ml were added and incubated for 30 min. For coculture: NCI-H358 orCalu-3 were plated into the wells containing fibroblast at a density of40,000 cells in 300 ul of NCI-H358 or Calu-3 media. 7 days later, mediawere collected and stored at −80 C for IL-6 detection. Cells were storedat −80 C until ready for RNA isolation and RT-PCR.

αSMA immunofluorescence staining: Cells were washed 2 times with PBS(200 ul in 96 well plates), and then fixed withFixation/Permeabilization Solution for 45 mins at room temperature (50uL in 96 well plate). Cell were then washed 3 times with 5 minincubation using 1×BD perm wash buffer (diluted in distilled water at1:10 dilution). The cells were blocked with Odyssey block buffer (50 ulin 96 well plates) for 60 min at room temperature. The plates werewashed 2 times with 5 min incubation between each wash using 1×BD permwash buffer (200 ul in 96 well plates). Anti-SMA antibody were added at1:100 dilution in wash buffer and incubated for 3 h at room temperature.Plates were then washed 2 times with 1×BD perm wash buffer (200 ul in 96well plate). Secondary Ab Goat Anti-mouse IgG conjugated with AlexaFluor 488 were used at 1:200 dilution in perm wash buffer in a finalvolume of 100 ul and incubated for 1 h at room temperature. Cells werewashed 2 times using 200 ul 1×BD perm wash buffer with 5 minutesincubation between each washes. DAPI were added to wash buffer at 1:1000dilution and 200 ul were added to the plated, incubated for 5 min atroom temperature, solution were aspirated and replaced with 200 ul of1×BD perm wash buffer.

For gene expression analysis. RNA were extracted according to the“RNeasy 96 Protocol for Isolation of Cytoplasmic RNA from AnimalCells-using Vacuum Technology” included in the RNeasy 96 Kit (Qiagen).cDNA Synthesis was done using the RT2 First Strand Kit and Real-Time PCRfor RT2 profiler PCR arrays with cycling conditions for AppliedBiosystems cyclers according to the procedures described in the RT2Profiler PCR Array Handbook (Qiagen).

Read-Outs Plate Reading Procedure for Imaging

Image Xpress Micro (IXM) machine and the MetaXpress software programwere used for image acquisition and analysis. For 2 color staining: DAPIand FITC staining, plate acquisition imaging protocol:“2014-YW-SMA488-DAPI-10x” were used. For data analysis, multi wavelengthcell scoring analysis parameter protocol “2014-5-7-YW-SMA488-DAPI-4x-2para a” were used to quantify the amount of SMA fiber induction due toFMT. Images of individual well were downloaded as BMP files.

IL-6 ELISA

Levels of human IL-6 were determined using a commercial IL-6 ELISA assay(Human Duoset IL-6, R & D Systems) following the manufacturer'sinstructions. Optical density reading (OD) at 450 nm are performed usingSpectramax M5e reader (Molecular Devices) and IL-6 concentration foreach sample extrapolated from a four-parameter logistic curve fitcalculated using OD reading from the internal IL-6 standard.

Gene Expression Analysis

After RT-PCR has completed the run in the QuantStudio 12k flex AppliedBiosystem cycler, CT values for all wells were downloaded to Excelspreadsheet for calculation of relative expression.

Computer Programs Used

Program Version Supplier Microsoft Office 2007/2013 Microsoft GraphPadPrism 5.02 GraphPad QuantStudio 12k flex Real time PCR 1.2.2 AppliedBiosystem system MetaXpress Image Analysis Software 3.1.0.97 MolecularDevices Adobe Photoshop CS6 13.0 X32 Adobe

Relative Gene Expression (Basal Level) and Fold Change Calculations

C_(T) Threshold cycle. The C_(T) is the cycle number at which thefluorescence generated within a reaction crosses the threshold line.C_(T) values are logarithmic and are used either directly forquantitative analyses.

For Relative Gene Expression (Basal Level):

Calculate the ΔC_(T) value. ΔC_(T)=C_(T target)−C_(T reference(GAPDH))

Calculate the 2^(−ΔCT) as relative gene expression at basal level.

For relative gene expression (fold change after treatment):

Calculate the ΔC_(T) value. ΔC_(T)=C_(T target)−C_(T reference(GAPDH))

Calculate the ΔΔC_(T) value. ΔΔC_(T)=ΔC_(T) testsample(treatment)−ΔC_(T) calibrator sample(without treatment)

The amount of target, normalized to an endogenous reference (e.g. GAPDH)and relative to a calibrator (before treatment or control), is given by:2^(−ΔΔCT)

Results Abituzumab Blocks Elevated αSMA Expression in H358-Fibroblastand Calu3-Fibroblast Co-Cultures (See Also FIG. 1)

The study produced substantial evidence for the hypothesized crosstalkbetween αv integrins and TGF-β during fibrotic processes. Tumorepithelial cell/fibroblast co-culture systems were used to mimic thepotential interaction of epithelial cells and fibroblasts in tissuesundergoing fibrosis. After 7 days of coculture basal level of aSMA werelow in lung fibroblast or dermal fibroblast mono-cultures. NCI-H358 andCalu-3 induced aSMA expression the fibroblast layer (FIG. 1). Additionof Abituzumab to NHLF+NCI-H358; NHLF+Calu-3; NHDF+NCI-H358; NHDF+Calu-3cocultures inhibited induction of aSMA expression in fibroblast.

Abituzumab Blocks Elevated Expression of FMT-Related Genes inH358-Fibroblast Co-Cultures (See Also FIG. 2)

Coculture of NHLF+NCI-H358; NHDF+NCI-H358; induced multiple mRNAtranscripts that are markers for FMT and also increased IL-6 production.In this system these markers were reduced by abituzumab treatment,demonstrating that αv integrins play a substantial role in FMT.

BIBLIOGRAPHY

-   Buscemi L, Ramonet D, Klingberg F, Formey A, Smith-Clerc J, Meister    J and Hinz B. The Single-Molecule Mechanics of the Latent TGF-β    Complex. Current Biology. 2009. 21: 2046-2054-   Eberlein C, Rooney C, Ross S J, Farren M, Weir H M, Barry S T.    E-Cadherin and EpCAM expression by NSCLC tumour cells associate with    normal fibroblast activation through a pathway initiated by integrin    αvβ6 and maintained through TGF-β signalling. Oncogene. 2015.    34:704-716.-   Eberlein C, Kendrew J, McDaid K, Alfred A, Kang J S, Jacobs V N,    Ross S J, Rooney C, Smith N R, Rinkenberger J, Cao A, Churchman A,    Marshall J F, Weir H M, Bedian V, Blakey D C, Foltz I N, Barry S T.    A human monoclonal antibody 264RAD targeting αvβ6 integrin reduces    tumour growth and metastasis, and modulates key biomarkers in vivo.    Oncogene. 2013. 32:4406-4416.-   Hata S, Okamura K, Haifa M, Ishikawa H, Yamazaki J. Proteolytic and    non-proteolytic activation of keratinocyte-derived latent TGF-β1    induces fibroblast differentiation in a wound-healing model using    rat skin. J Pharmacol Sci. 2014. 124: 230-243-   Mukhopadhyay A, Tan E K, Khoo Y T, Chan S Y, Lim I J, Phan T T.    Conditioned medium from keloid keratinocyte/keloid fibroblast    coculture induces contraction of fibroblast-populated collagen    lattices. Br J Dermatol. 2005. 152:639-645.-   Shephard P, Martin G, Smola-Hess S, Brunner G, Krieg T, Smola H.    Myofibroblast differentiation is induced in keratinocyte-fibroblast    co-cultures and is antagonistically regulated by endogenous    transforming growth factor-beta and interleukin-1. Am J    Pathol. 2004. 164:2055-2066.-   Renzoni E A, Abraham D J, Howat S, Shi-Wen X, Sestini P, Bou-Gharios    G, Wells A U, Veeraraghavan S, Nicholson A G, Denton C P, Leask A,    Pearson J D, Black C M, Welsh K I, du Bois R M. Gene expression    profiling reveals novel TGF-β targets in adult lung fibroblasts.    Respir Res. 2004. 5:24.-   Lygoe K A, Wall I, Stephens P, Lewis M P. Role of vitronectin and    fibronectin receptors in oral mucosal and dermal myofibroblast    differentiation. Biol Cell. 2007. 99:601-614-   Lygoe K A, Norman J T, Marshall J F, Lewis M P. AlphaV integrins    play an important role in myofibroblast differentiation. Wound    Repair Regen. 2004. 12:461-470.-   Gardner H, Strehlow D, Bradley L, Widom R, Farina A, de Fougerolles    A, Peyman J, Koteliansky V, Korn J H. Global expression analysis of    the fibroblast transcriptional response to TGF-β. Clin Exp    Rheumatol. 2004. 22(Suppl 33):S47-57

Example 2 Effect of Abituzumab on TGF-β Induced FMT in Human LungFibroblast Table of Figures

-   FIG. 3: TGF-β Increases Integrins Expression in Human Lung    Fibroblast-   FIG. 4: TGF-β Increases αSMA, IL-6 and other Myofibroblast Marker    Gene Expression in Lung Fibroblast-   FIG. 5: Abituzumab Treatment of Fibroblast Cultures Reduces the    TGF-β induced Increase in aSMA and IL-6-   FIG. 6: Abituzumab Treatment Reduces TGF-β-induced Collagen Gel    Contraction

List of Abbreviations αSMA: Alpha Smooth Muscle Actin BSA: Bovine SerumAlbumin Cy5: Cyanine 5 DAPI: 4′,6-Diamidino-2-Phenylindole ECM:Extracellular Matrix FGM: Fibroblast Growth Medium FMT: Fibroblast toMyofibroblast Transition FITC: Fluorescence Isothiocyanate IXM: ImageXpress Micro Screening System ug: Micgrogram mg: Milligram mL:Milliliter ng: Nanogram NHDF: Normal Human Dermal Fibroblasts NHLF:Normal Human Lung Fibroblasts PBS: Phosphate-Buffered Saline TGF-β1:Transforming Growth Factor-Beta 1 ELISAEnzyme-Linked Immunosorbent AssayFBS Fetal Bovine Serum h Human IL Interleukin Summary

TGF-β1 is shown here to be a potent mediator of fibroblast tomyofibroblast transition (FMT) which contributes to increasedextracellular matrix deposition and is main driver of fibrotic diseases.Furthermore, there is substantial evidence shown for crosstalk betweenaV integrins and TGF-β during these processes. TGF-β is secreted in alatent form which contains a Latency Associated Peptide (LAP) region.The LAP of TGF-β1 contains an RGD motif which interacts with theintegrins avβ1, avβ3, avβ5, avβ6 and avβ8 resulting in activation ofTGF-β1. Abituzumab is a human antibody specific for aV and thereforeinhibits avβ1, avβ3, avβ5, avβ6 and avβ8. This study shows anddetermines the effect of TGF-β on the aV integrin and fibrotic geneexpression and that abituzumab can block TGF-β induced gene expressionin vitro.

Expression of integrins was analyzed by RT-PCR and it was found thathuman lung fibroblasts express ITGB1>ITGB5>ITGB8>ITGB3. TGF-β-inducedFMT caused increased in the expression of ITGB5 and to a lesser extentITGB1 and ITGB3. TGF-β treatment increased myofibroblast marker genes inlung fibroblasts and immunofluorescence staining revealed increased inavβ5 expression. Abituzumab treatment reduced the increased expressionof aSMA, production of IL-6 and collagen gel contraction and thusdemonstrated an ability to block TGF-β induced FMT.

Introduction

Fibrotic diseases are characterized by excessive scarring due toproduction, deposition and contraction of extracellular matrix and isbelieved to be driven by myofibroblast proliferation and activation.Fibrotic diseases represent one of the largest groups of diseases forwhich there is no effective therapy. The fibrotic processes is regulatedby complex set of interactions within a network of profibrotic andantifibrotic mediators. TGF-β signaling is believed to play an importantrole in fibroblast to myofibroblast transition (FMT) which contributesto increased extracellular matrix deposition and is main driver ofdisease.

TGF-β isoforms are synthesized as latent precursors complexed withlatent TGF-β binding proteins, which contains a Latency AssociatedPeptide (LAP) region. There is substantial evidence for crosstalkbetween aV integrins and TGF-β during these processes. The LAP of TGF-β1contains an RGD motif which interacts with the integrins avβ1, avβ3,avβ5, avβ6 and avβ8 resulting in activation of TGF-β1. Abituzumab is apan-αv integrin antibody that binds allosterically to the ligand-bindingaV subunit and thus prevents ligand from binding to all αvβ heterodimersand therefore inhibits αv integrin-dependent activation of latent TGF-βand thus blocks acquisition of the myofibroblast phenotype byfibroblasts and other precursors. The present study provided strongevidence of the effect of TGF-β on the aV integrin and fibrotic geneexpression and that abituzumab blocks TGF-β induced gene expression innormal human lung fibroblasts (NHLF).

Materials and Methods Test Systems

The test system is the NHLF from healthy donors stimulated with TGF-β1with or without Latent TGF-β. Primary NHLF upregulate alpha smoothmuscle actin (primary readout) after TGF-β stimulation.

Description Company Cat # Lot# Tissue # Normal human lungLonza/clonetics CC-2512 0000374386 26789 fibroblasts Normal human lungLonza/clonetics CC-2512 0000343490 25745 fibroblasts Normal human lungLonza/clonetics CC-2512 0000369145 26646 fibroblasts

Test Material and Stimulus

Stimuli/Cytokine Company Cat # Lot# Recombinant human R & D Systems240-B-010 AV5513121 TGF-β 1 Recombinant human R & D Systems 299-LT-005FY1914031 TGF-β 1 latent Lot/ Antibodies Company Accession# Abituzumab(DI17E6) EMD Serono 402499 Anti-HEL IgG2 EMD Serono A12-145-2Antibodies/Dye for EMD Serono staining Company Lot # ID # αv Integrinantibody Merck KGaA 20100224 EM 013-09 αvB3 Integrin antibody Merck KGaA20091125 EM 277-03 αvB5 Integrin antibody Merck KGaA 20091125 EM 099-02αvB6 Integrin antibody Merck KGaA 20100224 EM 052-01 αvB8 Integrinantibody Merck KGaA 20100224 EM 133-09

Supplies and Instruments

Material/Reagent Provider Cat. # Lot # FGM Fibroblast Growth Lonza/CC-3132    354178 Medium and Bullet Kit clonetics (include Cat#CC-3131 &CC-4126) Fibroblast Growth Medium Lonza/ CC-3131    409065 cloneticsFibroblast Bullet Kit Lonza/ CC-4126    417247 cloneticsGentamicin-Sulfate Lonza/ CC-4081J    417245 clonetics Insulin Lonza/CC-4021J    417243 clonetics rhFGF Lonza/ CC-4065J    417244 cloneticsFBS Lonza/ CC-4101J    417246 clonetics Reagent Pack/Subculture Lonza/CC-5034    409088 Reagents clonetics Hepes Buffered Saline Lonza/CC-5022    362157 clonetics Trypsin Lonza/ CC-5012    372708 cloneticsTrypsin Neutralizing Lonza/ CC-5002    385562 Solution clonetics T 150cm2 flasks Corning 430825  19514018 UltraPure Distilled Water Invitrogen10977-015   1607416 PBS Invitrogen 20012-027   1457916 Collagen 1 Cellware BD 356700   2136233 96-well Black/Clear Plate Biosciences 24 wellplates Falcon 353047 N/A Odyssey Blocking Buffer Li-COR 927-40000 T1752(500 mL) BD Cytofix/Cytoperm BD 554714   3099684Fixation/Permeabilization Biosciences kit Fixation Solution BD 554722  3231840 Biosciences Permeabilization (Perm) BD 554723   4174605 WashBuffer Biosciences Anti-actin, smooth muscle, Millipore CBL171   2377949clone ASM-1 Goat Anti-mouse IgG Alexa Invitrogen A11001   1503602Fluor488: Invitrogen Corp Goat Anti-rabbit IgG Alexa Invitrogen A21245  1623067 Fluor647: Invitrogen Corp DAPI, dilactate Sigma D9564-042M4005 Aldrich 10MG RNeasy 96 kit Qiagen 74181 148019008 RT2 FirstStrand kit Qiagen 330401 N/A RT2 Profiler PCR Array Qiagen 12473 N/A(Custom) Cell Contraction assay kit Cell CBA-201 N/A BioLabs, IncEquipment: Company Serial # Allegra X-14R Centrifuge Beckman CoulterB34604-AA Laminar hood model 1395, Thermo Fisher 114627-128 1300SeriesA2 Incubator at 37 C. with Thermo Fisher 313910-5441 humidified 5%CO2 Spectramax M5e Molecular MVE06101 Devices Biotek 405LS microplatePerkin Elmer 1306126 washer Microscope, Nikon TMS DSC Optical 11345Services Water Bath Equipnet 35933-2-2 Image Xpress Micro Molecular12264 Imaging Microscope Devices Optiplex 990 Image Dell X16-96076Analysis Computer QuantStudio 12K Flex Applied 285880466 Biosystems byLife Technologies

Design Outline Study Design

NHLF culture media (FGM with 2% FBS): FGM™-2 BulletKit™ (CC-3132)contains one 500 ml bottle of Fibroblast Cell Basal Medium (CC-3131) andFibroblast Bullet Kit (CC-4126) with the following growth supplements:0.5 ml hFGF-B (CC-4065J); 0.5 ml Insulin (CC-4021J); 10 ml FBS(CC-4101J); 0.5 ml GA-1000 (CC-4081J).

NHLF culture media for FMT assays (FGM with 0.1% FBS): FGM™-2 BulletKit™(CC-3132) contains one 500 ml bottle of Fibroblast Cell Basal Medium(CC-3131) and Fibroblast Bullet Kit (CC-4126) with the following growthsupplements: 0.5 ml hFGF-B (CC-4065J); 0.5 ml Insulin (CC-4021J); 0.5 mlFBS (CC-4101J); 0.5 ml GA-1000 (CC-4081J).

NHLF were grown in T150 tissue culture flasks in FGM with 2% FBS untilthey were 70-80% confluent on the day of the assay. The cells wererinsed with 6 ml HEPES buffered saline solution (Lonza Cat#CC-5022),trypsinized with 6 ml trypsin/EDTA (Lonza Cat#CC-5012) for 5 min at roomtemperature. The trypsin was inactivated with 6 ml trypsin neutralizingsolution (Lonza Cat#CC-5002), spun down at 400 g for 4 min and washedonce with FGM with 2% FBS. For imaging studies, cells were seeded at7500 cells/well in Collagen 1 Cellware 96-well Black/Clear Plate. Forgene expression studies, cells were seeded at 50,000 cells/well in 24wells culture plates.

Cells were cultured for 8 hours in FGM with 2% FBS. Media were aspiratedand cells were starved overnight in FGM containing 0.1% FBS.

To investigate the effect of TGF-β on the aV integrins, aSMA and geneexpression: Media were aspirated and 100 ul of FGM containing 0.1% FBSwere added, TGF-β were added at 2× (20 ng/ml) of desired concentrationin 100 ul of FGM containing 0.1% FBS. 72 hours later, media werecollected and stored at −80 C for IL-6 detection.

To investigate the effect of Abituzumab on TGF-β induced fibroblastmyofibroblast transition (FMT), NHLF were seeded at 7500 cells/well inCollagen 1 Cellware 96-well Black/Clear Plate, cultured for 8 hours inFGM with 2% FBS. Media were aspirated and cells were starved overnightin FGM containing 0.1% FBS. Media were aspirated and 100 ul of FGM with0.1% FBS containing abituzumab or anti-HEL IgG at a conc of 25 ng/mlwere added and incubated for 30 min. TGF-β were added at 2× (LatentTGF-β1=40 ng/ml, TGF-β=0.312 ng/ml) of desired concentration in 100 ulof FGM containing 0.1% FBS. 72 hours later, media were collected andstored at −80 C for IL-6 detection.

For aV integrins, aSMA expression studies using immunofluorescence,cells were fixed and stained according to the alpha smooth muscle actin(αSMA) and aV Integrin staining procedure. For FMT related geneexpression analysis, cells were stored at −80 C until ready for RNAisolation and RT-PCR.

Alpha smooth muscle actin and aV Integrins immunofluorescence staining:Cells were washed 2 times with PBS (200 ul in 96 well plates), and thenfixed with Fixation/Permeabilization Solution for 45 mins at roomtemperature (50 uL in 96 well plate). Cell were then washed 3 times with5 min incubation using 1×BD perm wash buffer (diluted in distilled waterat 1:10 dilution). The cells were blocked with Odyssey block buffer (50ul in 96 well plates) for 60 min at room temperature. The plates werewashed 2 times with 5 min incubation between each wash using 1×BD permwash buffer (200 ul in 96 well plates). Anti-SMA antibody were added at1:100 dilution in wash buffer. Anti-Integrin Ab were used at 1 ug/ml inperm buffer as final concentration and incubated for 3 h at roomtemperature. Plates were then washed 2 times with 1×BD perm wash buffer(200 ul in 96 well plate). Secondary Ab Goat Anti-mouse IgG conjugatedwith Alexa Fluor 488 were used at 1:200 dilution and Secondary Ab GoatAnti-rabbit IgG conjugated with Alexa Fluor 647 were used at 1:100dilution in perm wash buffer in a final volume of 100 ul and incubatedfor 1 h at room temperature. Cells were washed 2 times using 200 ul 1×BDperm wash buffer with 5 minutes incubation between each washes. DAPIwere added to wash buffer at 1:1000 dilution and 200 ul were added tothe plated, incubated for 5 min at room temperature, solution wereaspirated and replaced with 200 ul of 1×BD perm wash buffer.

For gene expression analysis. RNA were extracted according to the“RNeasy 96 Protocol for Isolation of Cytoplasmic RNA from AnimalCells-using Vacuum Technology” included in the RNeasy 96 Kit (Qiagen).cDNA Synthesis was done using the RT2 First Strand Kit and Real-Time PCRfor RT2 profiler PCR arrays with cycling conditions for AppliedBiosystems cyclers according to the procedures described in the RT2Profiler PCR Array Handbook (Qiagen).

For gel contraction assay: Human Fibroblasts grown in T150 were rinsedonce with HEPES and supernatants were removed and 6 ml of trypsin wereadded to each flask for 5 min at room temperature, 6 ml of trypsinneutralizing solution were added and cells were collected bycentrifugation at 400 g. Cells were resuspended in FGM with 2% FBS andat 2×106 cells/ml. Cells were treated with 10 ug/ml Abituzumab oranti-HELIgG for 15 mins. Collagen lattice were formed by mixing 2 parts(120 ul) of ‘treated’ cell suspension and 8 parts (480 ul) of coldcollagen gel solution with or without TGF-β (10 ng/ml). 500 ul of thecell-collagen mixture were transferred to each well in a 24 well plate,incubate 1 h at 37 C. After collagen polymerization, 1 ml of culturemedium was added on top of each collagen gel lattice. Collagen gel isgently lifted up using a sterile spatula, the gel is allowed to float inthe media. Collagen gel size was monitored for 5 days and pictures weretaken on day 5.

Read-Outs Plate Reading Procedure for Imaging

Image Xpress Micro (IXM) machine and the MetaXpress software programwere used for image acquisition and analysis. For 3 color staining:DAPI, FITC, and Cy5, plate acquisition imaging protocol:“2014-YW-SMA-Int-DAPI-647-10x” were used. For 2 color staining: DAPI andFITC staining, plate acquisition imaging protocol:“2014-YW-SMA488-DAPI-10x” were used. For data analysis, multi wavelengthcell scoring analysis parameter protocol “2014-5-7-YW-SMA488-DAPI-4x-2para a” were used to quantify the amount of SMA fiber induction due toFMT. Images of individual well were downloaded as BMP files.

IL-6 ELISA

Levels of human IL-6 were determined using a commercial IL-6 ELISA assay(Human Duoset IL-6, R & D Systems) following the manufacturer'sinstructions. Optical density reading (OD) at 450 nm are performed usingSpectramax M5e reader (Molecular Devices) and IL-6 concentration foreach sample extrapolated from a four-parameter logistic curve fitcalculated using OD reading from the internal IL-6 standard.

Gene Expression Analysis

After RT-PCR has completed the run in the QuantStudio 12k flex AppliedBiosystem cycler, CT values for all wells were downloaded to Excelspreadsheet for calculation of relative expression.

Computer Programs Used

Program Version Supplier Microsoft Office 2007/2013 Microsoft GraphPadPrism 5.02 GraphPad QuantStudio 12k flex Real time PCR 1.2.2 AppliedBiosystem system MetaXpress Image Analysis Software 3.1.0.97 MolecularDevices Adobe Photoshop CS6 13.0 X32 Adobe

Relative Gene Expression (Basal Level) and Fold Change Calculations

C_(T) Threshold cycle. The C_(T) is the cycle number at which thefluorescence generated within a reaction crosses the threshold line.C_(T) values are logarithmic and are used either directly forquantitative analyses.

For Relative Gene Expression (Basal Level):

Calculate the ΔC_(T) value. ΔC_(T)=C_(T target)−C_(T reference(GAPDH))

Calculate the 2^(−ΔCT) as relative gene expression at basal level.

For relative gene expression (fold change after treatment):

Calculate the ΔC_(T) value. ΔC_(T)=C_(T target)−C_(T reference(GAPDH))

Calculate the ΔΔC_(T) value. ΔΔC_(T)=ΔC_(T) testsample(treatment)−ΔC_(T) calibrator sample(without treatment)

The amount of target, normalized to an endogenous reference (e.g. GAPDH)and relative to a calibrator (before treatment or control), is given by:2^(−ΔΔCT)

Results TGF-β Increases Integrins Expression in Human Lung Fibroblast(See Also FIG. 3)

Expression of integrins in NHLF were analyzed by RT-PCR and it was foundthat NHLFs express integrins at steady state and expression level in theorder ITGB1>ITGB5>ITGAV>ITGB8>ITGB3. It has been hypothesized that thereis crosstalk between aV integrins and TGF-β, therefore, we looked at theeffect of TGF-β in Integrins expression in FMT assays. We found thatTGF-β-induced FMT caused increased in the expression of ITGB5 and to alesser extent ITGB1 and ITGB3; Immunofluorescence staining revealedincreased in αvβ5 expression (see FIG. 3: TGF-β Increases IntegrinsExpression in Human Lung Fibroblast)

TGF-β Increases aSMA, IL-6 and Other Myofibroblast Marker GeneExpression in Lung Fibroblast (See Also FIG. 4)

TGF-β treatment increased IL6 production and alpha smooth muscleexpression as detected by immunofluorescence and RT-PCR. TGF-β treatmentincreased myofibroblast marker gene such as alpha smooth muscle,collagen, fibronectin, SERPINE1, SNAI1, periostin, N-Caherin expressionin lung fibroblasts (see FIG. 4: TGF-β Increases aSMA, IL-6 and otherMyofibroblast Marker Gene Expression in Lung Fibroblast).

Abituzumab Treatment of Fibroblast Cultures Reduces the TGF-β-InducedIncrease in aSMA and IL-6 (See Also FIG. 5)

As shown in FIG. 1, TGF-β increase expression of Integrins, in thisexperiments we treated NHLF with suboptimal dose of active TGF-β toincrease expression of integrin together with height dose of Latent formTGF-β. Combination of Latent and active TGF-β1 induced higher number ofcells that expressed aSMA compared to Latent or active form alone.Abituzumab treatment reduced the expression of aSMA, production of IL-6and FMT gene expression caused by TGF-β activation (see FIG. 5:Abituzumab Treatment of Fibroblast Cultures Reduces the TGF-β-inducedIncrease in aSMA and IL-6).

Abituzumab Treatment Reduces TGF-β-Induced Collagen Gel Contraction (Seealso FIG. 6)

3 dimensional collagen gel assays were used in fibroblast contractionstudies. To determine whether the changes in aSMA protein by TGF-β1 andblockade of aV integrins but Abituzumab had a functional consequence,collagen gel contraction assays were performed. When Abituzumab wasadded to the cells, it reduced the degree of contraction caused by theaddition of TGF-β (see FIG. 6: Abituzumab Treatment ReducesTGF-β-induced Collagen Gel Contraction).

Discussion

In this report, we showed that TGF-β increases aV integrins, aSMA, andother FMT genes such as collagen, fibronectin, SERPINE1, SNAI1,periostin, N-Caherin expression in human lung fibroblast. Abituzumabtreatment of fibroblast cultures reduces the TGF-β-induced increases inaSMA, IL6, CTGF, SERPINE, PLOD.

Abituzumab is been shown to block αv integrin-dependent activation oflatent TGF-β at sites of extracellular matrix overproduction. Here weshowed, using in vitro culture systems, abituzumab blocks fibroblastmyofibroblast transition. TGF-β upregulates aV integrins, blocking αvIntegrin-dependent activation of latent TGF-β, blocks local upregulationof integrins on myofibroblast cell membranes, and thus is able to breakthe vicious cycle of TGF-β activation and myofibroblast accumulation.

-   Literature-   Buscemi L, Ramonet D, Klingberg F, Formey A, Smith-Clerc J, Meister    J and Hinz B. The Single-Molecule Mechanics of the Latent TGF-β    Complex. Current Biology. 2009. 21: 2046-2054-   Goodman S L, Grote H J, Wilm C. Matched rabbit monoclonal antibodies    against αv-series integrins reveal a novel αvβ3-LIBS epitope, and    permit routine staining of archival paraffin samples of human    tumors. Biol Open. 2012. 1(4):329-340.-   Scaffidi A K, Petrovic N, Moodley Y P, Fogel-Petrovic M, Kroeger K    M, Seeber R M, Eidne K A, Thompson P J, Knight D A. alpha(v)beta(3)    Integrin interacts with the transforming growth factor beta (TGF-β)    type II receptor to potentiate the proliferative effects of TGF-β1    in living human lung fibroblasts. J Biol Chem. 2004.    279(36):37726-37733-   Renzoni E A, Abraham D J, Howat S, Shi-Wen X, Sestini P, Bou-Gharios    G, Wells A U, Veeraraghavan S, Nicholson A G, Denton C P, Leask A,    Pearson J D, Black C M, Welsh K I, du Bois R M. Gene expression    profiling reveals novel TGF-β targets in adult lung fibroblasts.    Respir Res. 2004. 5:24.-   Lygoe K A, Wall I, Stephens P, Lewis M P. Role of vitronectin and    fibronectin receptors in oral mucosal and dermal myofibroblast    differentiation. Biol Cell. 2007. 99(11):601-614-   Lygoe K A, Norman J T, Marshall J F, Lewis M P. AlphaV integrins    play an important role in myofibroblast differentiation. Wound    Repair Regen. 2004. 12(4):461-470.-   Gardner H, Strehlow D, Bradley L, Widom R, Farina A, de Fougerolles    A, Peyman J, Koteliansky V, Korn J H. Global expression analysis of    the fibroblast transcriptional response to TGF-β. Clin Exp    Rheumatol. 2004. 22(Suppl 33):S47-57

Appendix Gene List for RT-PCR

RT2 Gene Catalog Symbol Alias Refseq # Official Full Name Number ITGAVCD51/MSK8/VNRA/VTNR NM_002210 Integrin, alpha V PPH00628 (vitronectinreceptor, alpha polypeptide, antigen CD51) ITGB1 CD29/FNRB/GPIIA/MDF2/NM_002211 Integrin, beta 1 PPH00650 MSK12/VLA-BETA/VLAB (fibronectinreceptor, beta polypeptide, antigen CD29 includes MDF2, MSK12) ITGB3BDPLT16/BDPLT2/CD61/ NM_000212 Integrin, beta 3 PPH00178 GP3A/GPIIIa/GT(platelet glycoprotein IIIa, antigen CD61) ITGB5 — NM_002213 Integrin,beta 5 PPH00634 ITGB6 — NM_000888 Integrin, beta 6 PPH00630 ITGB8 —NM_002214 Integrin, beta 8 PPH00647 POSTN OSF-2/OSF2/PDLPOSTN/ NM_006475Periostin, PPH12343 PN/periostin osteoblast specific factor TNC150-225/DFNA56/GMEM/GP/ NM_002160 Tenascin C PPH02442 HXB/JI/TN/TN-C VTNV75/VN/VNT NM_000638 Vitronectin PPH00253 CDH1 Arc-1/CD324/CDHE/ECAD/NM_004360 Cadherin 1, PPH00135 LCAM/UVO type 1, E-cadherin (epithelial)CDH2 CD325/CDHN/CDw325/ NM_001792 Cadherin 2, type 1, PPH00636 NCADN-cadherin (neuronal) CLDN1 CLD1/ILVASC/SEMP1 NM_021101 Claudin 1PPH02779 OCLN BLCPMG NM_002538 Occludin PPH02571 TJP1 ZO-1 NM_175610Tight junction PPH09919 protein 1 (zona occludens 1) VIM CTRCT30/HEL113NM_003380 Vimentin PPH00417 KRT5 CK5/DDD/DDD1/EBS2/K5/ NM_000424 Keratin5 PPH02625 KRT5A CTNNB1 CTNNB/MRD19/armadillo NM_001904 Catenin(cadherin- PPH00643 associated protein), beta 1, 88 kDa ACTA2AAT6/ACTSA/MYMY5 NM_001613 Actin, alpha 2, PPH01300 smooth muscle, aortaCOL1A1 OI4 NM_000088 Collagen, type I, PPH01299 alpha 1 COL1A2 OI4NM_000089 Collagen, type I, PPH01918 alpha 2 FN1 CIG/ED-B/FINC/FN/FNZ/NM_002026 Fibronectin 1 PPH00143 GFND/GFND2/LETS/MSF KLF4 EZF/GKLFNM_004235 Kruppel-like factor PPH18388 4 (gut) ZEB1 AREB6/BZP/DELTAEF1/NM_030751 Zinc finger E-box PPH01922 FECD6/NIL2A/PPCD3/TCF8/ bindinghomeobox ZFHEP/ZFHX1A 1 Gene Symbol Alias Refseq # Official Full NameCCL2 GDCF-2/HC11/HSMCR30/ NM_002982 Chemokine (C-C motif)MCAF/MCP-1/MCP1/ ligand 2 SCYA2/SMC-CF CCL3 G0S19-1/LD78ALPHA/ NM_002983Chemokine (C-C motif) MIP-1-alpha/MIP1A/SCYA3 ligand 3 SERPINE1PAI/PAI-1/PAI1/PLANH1 NM_000602 Serpin peptidase inhibitor, clade E(nexin, plasminogen activator inhibitor type 1), member 1 SNAI1SLUGH2/SNA/SNAH/ NM_005985 Snail homolog 1 SNAIL/SNAIL1/dJ710H13.1(Drosophila) ACTB BRWS1/PS1TP5BP1 NM_001101 Actin, beta GAPDH G3PD/GAPDNM_002046 Glyceraldehyde-3- phosphate dehydrogenase HGDC HIGX1A SA_00105Human Genomic DNA Contamination RTC RTC SA_00104 Reverse TranscriptionControl PPC PPC SA_00103 Positive PCR Control PLOD2 LH2/TLH NM_182943Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 CTGFCCN2/HCS24/IGFBP8/ NM_001901 Connective tissue NOV2 growth factor

Example 3 Effect of Abituzumab on Binding of αvβ6 to Latency-AssociatedPeptide (LAP) List of Abbreviations αSMA: Alpha Smooth Muscle Actin ECM:Extracellular Matrix ug: Microgram mg: Milligram mL: Millilitre

LAP: latency associated peptide (LAP)LTBP: latent TGF-β binding protein

ng: Nanogram TGF-β1: Transforming Growth Factor-Beta 1 ELISA:Enzyme-Linked Immunosorbent Assay h: Human IL: Interleukin

Summary αvβ3, αvβ3, αvβ6 and αvβ8 can Control the Activation of TGF-β

TGF-β1 is a major pro-fibrotic cytokine that plays an important part inthe development of tissue fibrosis in various organs. TGF-β 1contributes to fibrosis by promoting the differentiation of tissuefibroblasts into myofibroblasts. Myofibroblasts can produceextracellular matrix (ECM) proteins, and are the main drivers for ECMexpansion and contraction during fibrosis. TGF-β1 is secreted in alatent complex, which in addition to TGF-β 1, also contains a latencyassociated peptide (LAP) and latent TGF-β binding protein (LTBP). Thecomplex can adopt an open conformation to release active TGF-β1 whenLTBP is anchored to ECM and LAP binds one of the αv integrins, namelyαvβ1, αvβ3, αvβ5, αvβ6 and αvβ8, on the αv subunit.

Abituzumab is a human antibody specific for the ligand-binding αvsubunit of αv integrins. Upon binding to the αv subunit, abituzumab canprevent αv integrins from binding to its ligands such as LAP. The goalof this study was to evaluate the ability of abituzumab to block bindingof LAP to αvβ6.

Using an anti-αvβ6-based ELISA assay, we demonstrated that abituzumabwas able to block the binding of αvβ6 to LAP precoated to ELISA platesin a dose-dependent manner. The finding clearly demonstrate the abilityof abituzumab to block binding of αv integrins to LAP, a key step in theactivation of TGF-β. This forms part of the scientific basis fordeveloping abituzumab for treating fibrotic diseases.

Introduction

TGF-β1, a major pro-fibrotic cytokine, plays an important part in thedevelopment of tissue fibrosis in various organs. TGF-β1 contributes tofibrosis by promoting the differentiation of tissue fibroblasts intomyofibroblasts. Myofibroblasts can produce extracellular matrix (ECM)proteins and contract. They are the main drivers for ECM expansion andcontraction during fibrosis. TGF-β1 is secreted in a latent complexwhich in addition to TGF-β 1, also contains a latency associated peptide(LAP) and latent TGF-β binding protein (LTBP). The complex can adopt anopen conformation to release active TGF-β1 when LTBP is anchored to ECMand LAP binds one of the αv integrins, namely αvβ1, αvβ3, αvβ5, αvβ6 andαvβ8, on the αv subunit.

The purpose of the present study was to evaluate the ability ofabituzumab to block binding of LAP to αvβ6.

Materials and Methods Materials Test Systems

The test system is a competitive binding assay in which a plate iscoated with recombinant human LAP and then incubated with recombinanthuman αvβ6 in the presence or absence of abituzumab also termed DI17E6.The amount of αvβ6 bound to LAP is detected by ELISA using a rabbit antiαvβ6 antibody (capture antibody) and a goat anti-rabbit IgG (detectingantibody) conjugated with horse radish paraoxide (HRP).

Materials and Instruments Key Materials

Catalog/internal Abbreviated name Origin code # Lot # αvβ6 integrin Inhouse 262Y08S1.INT Pool B DI17E6 In house EMD 525797 40350 DI17E6 Inhouse EMD 525797 13245 LAP R&D 246-LP-025/CP IK2613061 Systems 17E6 Inhouse 265Y08C2.G01 265Y08C2.G01/ Pool A anti-αvβ6 antibody in houseMSB0011521H 1 Anti-HEL IgG in house MSB0011523H 1 425(VL)/17E6(VH) Inhouse NA A-10-118-2 g1 hinge IgG2 (FN->AQ) anti-αvβ6 antibody MilliporeMab2077Z 2430505 10D5 anti-αvβ6 antibody In house NA EMD84266′-02 LM609Rabbit anti-αvβ6 In house NA EMD05201-1: #1/b: #1 Goat anti-rabbitBiorad 172-1019 LS1/21019 IgG ELISA plates Greiner 07230103 NA

Key Equipment

EMD Serono Equipment: Company ID # Serial # ELISA plate reader TecanELISA 70160370 911007538 Reader InfiniteM200

For other materials and equipment, see report SWE00174.

Coating Buffer (in Double Distilled Water): 200 mM Tris 150 mM NaCl

Adjust pH to 7.4 with HCl

+1 mM CaCl2); 1 mM MgCl2; 0.01 mM MnCl2 Blocking Buffer (in DoubleDistilled Water): 50 mM Tris 100 mM NaCl

Adjust pH to 7.4 with HCl+5 mg/ml BSA

Wash and Dilution Buffer (in Double Distilled Water): 200 mM Tris 150 mMNaCl

Adjust pH to 7.4 with HCl

+1 mM CaCl2); 1 mM MgCl2; 0.01 mM MnCl2

+0.1 mg/kg BSA

Experimental Design Outline Study Design

Coating of Plates with LAP.

Recombinant human. LAP was added to a plate at a final concentration of0.1 μg/ml and incubated overnight at 4° C. for coating the plate withLAP. The plate was then drained and incubated with 100 μl of blockingbuffer for 2 hours at 37° C. to block non-specific binding of αvβ6 tothe plates.

Addition αvβ6 and Abituzumab or Control Antibodies to LAP Coated Plates.

50 μl of pre-diluted abituzumab or control antibodies were added to eachwell from high to low concentrations in a serial dilution. Then 50 μlthe αvβ6 solution was added to each well. The final concentrations forthe antibodies were 2.5 μg/ml-0.16 ng/ml, and 0.25 μg/ml for αvβ6.

The effect of abituzumab was compared with two other anti-αvβ6antibodies: MSB0011521H-1 and 10D5. IgG2a was used as isotype controlIgG for abituzumab. Anti-HEL (MSB0011523H-1) is an antibody specific forthe non-mammalian protein Hevein-like preproprotein from Arabidopsis,and was used as a negative control antibody for MSB0011521H-1. 17E6 isthe original non-deimmunized mouse pan anti-αv antibody from whichabituzumab or DI17E6 was derived. These antibodies were added at thesame concentrations as abituzumab or DI17E6.

The plate was incubated for 60 min at 37° C. with αvβ6, and abituzumabor its control antibodies. The plates were then washed for addition ofsecondary antibodies.

Addition HRP-Conjugated Goat Anti-Rabbit IgG to Plates, ColorDevelopment and Reading.

The plate was added HRP-conjugated goat anti-rabbit IgG and incubatedfor 90 min at 37° C. The plate was then added R&D substrate reagent(Pack DY999) and incubated for 20 min to develop the color before thereaction was stopped with the addition of R&D stop solution DY994. Theplate was then read at 450 nm on the Tecan ELISA Reader InfiniteM200.

Read-Outs

Binding of αvβ6 to LAP Measured by Using ELISA.

The amount of αvβ6 bound to LAP was determined by using the proceduresdescribed above in Study Design.

Results

Abituzumab Inhibits Binding of αvβ6 to LAP (See Also FIG. 7)

Binding of αvβ6 to precoated LAP was detected by a specific anti αvβ6antibody-based ELISA. The binding was blocked in aconcentration-dependent manner by DI17E6 when it was co-incubated withαvβ6 in a plate precoated with LAP. The anti-αvβ6 antibodiesMSB0011521H-1 and 10D5 also inhibited the binding. In contrast, neitherthe isotype control IgG2 nor the negative control antibody (anti-HEL,MSB0011523H-1) displayed any inhibitory effects. Also the αvβ3 integrinspecific inhibitory antibody LM609 did not inhibit αvβ6 binding to LAP.The findings indicate that DI17E6 specifically inhibit the binding ofαvβ6 to LAP.

Binding of αvβ6 to LAP Measured by Using ELISA.

The amount of αvβ6 bound to LAP was determined by using the proceduresdescribed above in Study Design.

Results

Abituzumab Inhibits Binding of αvβ6 to LAP

Binding of αvβ6 to precoated LAP was detected by a specific anti αvβ6antibody-based ELISA. The binding was blocked in aconcentration-dependent manner by DI17E6 when it was co-incubated withαvβ6 in a plate precoated with LAP. The anti-αvβ6 antibodiesMSB0011521H-1 and 10D5 also inhibited the binding. In contrast, neitherthe isotype control IgG2 nor the negative control antibody (anti-HEL,MSB0011523H-1) displayed any inhibitory effects. Also the αvβ3 integrinspecific inhibitory antibody LM609 did not inhibit αvβ6 binding to LAP.The findings indicate that DI17E6 specifically inhibit the binding ofαvβ6 to LAP.

Tabulated Study Report

Title of study: Effect of abituzumab on Binding of avβ6 toLatency-Associated Peptide (LAP) Principle of test: Competitive bindingELISA assay Biological materials avβ6 Experimental Binding of avβ6 topre-coated LAP/co- conditions/treatment incubation of avβ6 andabituzumab for schedule: competitive inhibition Test Article: AbituzumabTreatment of controls: Anti-HEL (MSB0011523H-1, negative control), IgG2a(isotype control) Method of evaluation/ Precentage of avβ6 binding toLAP detected by Endpoints: avβ6 ELISA, with blank (no avβ6) as 0% andavβ6 alone as 100%. Results: Abituzumab inhibited avβ6 binding to LAP ina concentration-dependent manner. Conclusions: Abituzumab specificallyinhibits avβ6 binding to LAP in a concentration dependant manner

Discussion

In this study, we showed that abituzumab (DI17E6) specifically andconcentration-dependently inhibited αvβ6 binding to LAP. The findingclearly demonstrate the ability of abituzumab to block binding of αvintegrins to LAP, a key step in the activation of TGF-β. This evidenceforms an important part of the scientific basis for using abituzumab fortreating fibrosis and/or fibrotic diseases (see also FIG. 7: FIG. 7shows the inhibition of αvβ6 binding to LAP by Abituzumab). Binding ofαvβ6 to coated LAP was detected as an increase in the optical density inwells containing αvβ6 (untreated) over the blank (buffer with no αvβ6).The values were normalized with the former as a 100% and the latter as0%. Both batches of DI17E6 (batch #40350 and 13245) reduced the bindingof αvβ6 to LAP in a concentration-dependent manner, similarly to theanti-αvβ6 antibody MSB0011521H-1, but better than anti-αvβ6 antibody10D5. In contrast, neither the isotype control IgG (IgG2) nor thenegative control antibody (anti-HEL, MSB0011523H-1, data not shown)affected the binding at the same concentrations range. Also the αvβ3specific inhibitory antibody LM609 did not inhibit αvβ6 binding to LAP.

Example 4 Fibrosis/Systemic Sclerosis Gene Signature Summary

-   -   The goal of this study was to find a robust gene signature for        diagnosing & monitoring the status of fibrosis in patients with        systemic sclerosis (SSc).    -   Genes for the signature were identified using a dual strategy:        -   A list of literature-based genes was pre-defined and tested            for up-regulation in SSc skin in published microarray-based            gene expression data.        -   In an unbiased analysis of all genes, three public SSc data            sets were used to find candidate genes up-regulated in SSc            skin.    -   Further, the pre-final candidate gene list was filtered on two        public genome-scale gene expression data sets for their        association with pulmonary fibrosis.    -   Finally, the list of candidate genes was filtered for        up-regulation in immune-related tissues compared to solid        tissues.    -   In summary, we were able to find a robust signature of 19        non-immune-related genes up-regulated in SSc and pulmonary        fibrotic tissue by analyzing five appropriate data sets.    -   In an experiment with human fibroblasts that were either        supplemented with TGF-β or co-cultured with cells from the human        epithelial H358 lung cell line a gene to be included in the        TGF-β-up/Abituzumab-down signature (in the following termed TUAD        signature) had to show an up-regulation after administration of        TGF-β (median fold change across 3 repeated experiments).    -   Further, the final TUAD signature of 9 genes was subjected to an        experiment with normal human lung fibroblast (NHFL) that were        co-cultured with human H358 cells and treated either with        anti-HEL (hen egg lysozyme) or abituzumab at two doses. The TUAD        was to be accepted as TGF-β linked fibrosis signature that can        be down-modulated by Abituzumab, because a) of a down-regulation        of all genes in the 9-gene signature after Abituzumab treatment,        and b) because of the down-regulation of the net TUAD 9-gene        signature score.

Introduction

This study aims to was to find a robust gene signature for monitoring offibrosis in patients with systemic sclerosis (SSc) using a two-tierprocess. Literature-based genes reported to be up-regulated in SSc andTGF-simulated fibroblasts as well as genes with strong evidence ofup-regulation in patients with SSc/fibrosis in public data sets were tobe subjected to further analysis to finally yield a robust genesignature for fibrosis in SSc and IPF.

In addition, we performed nanostring expression profiling experiments toidentify another signature, termed TUAD signature, of genes that areup-regulated by TGF-β, thus representing a TGF-β dependent gene subgroupof the 19 gene signature. Finally, we subjected this TUAD signature toanother experiment to test whether Abituzumab can down-regulate the TUADsignature.

Materials

For the assessment of up-regulated genes in SSc/fibrosis we use thefollowing data sets. Data sets are available in the Gene ExpressionOmnibus (GEO) data base.

Gene expression is obtained by averaging probe set intensities per gene.

Systemic Sclerosis Data Sets

GSE9285 (Milano et al., 2008) contains 75 samples in total. Skinbiopsies were taken from the forearm and back of 17 patients withdiffuse SSc (dSSc), 7 with limited SSc (ISSc), 3 patients with morpheaand 6 healthy controls.

Data set GSE32413 (Pendergrass et al., 2012) consists of 89 skin biopsysamples in total. Gene expression was measured of 22 patients with SScand 9 healthy controls. 13 of the SSc patients were treated withrituximab, 9 were untreated. We use the baseline/pre-treatment samplesfor the assessment of differential expression between SSc skin andnormal skin and exclude the biopsies taken 6 and 36 months aftertreatment.

GSE45485 (Hincliff et al., 2013) contains 83 skin biopsies in total from12 SSc patients treated with mycophenolate motefil (MMF) and 10 healthycontrols. Only baseline (pre-treatment) samples from SSc patients wereincluded in the analysis, samples taken 6 and 12 months after MMFtreatment were excluded.

Pulmonary Fibrosis Data Sets

GSE24206 (Meltzer et al., 2011) 17 lung samples from 11 patients withearly/advanced Idiopathic Pulmonary Fibrosis (IPF). 6 patients provideda pair of samples from upper and lower lobes, 5 patients contributedsingleton samples). 6 control specimens were obtained from routine lungvolume reduction of healthy donor lungs at the time of lungtransplantation.

Data set GSE48149 (unpublished) contains 53 lung samples in total. 8patients with Idiopathic Pulmonary Arterial Hypertension (IPAH), 13patients with IPF, 10 patients with Pulmonary Arterial Hypertension dueto SSc (SSc-PAH) and 13 patients with Pulmonary Fibrosis due to SSc(SSc-PF) are included as well as 9 healthy controls. GSE21369 comprises29 lung tissue samples of 23 subjects. Data of 11 patients diagnosedwith Usual Interstitial Pneumonia/Idiopathic Pulmonary Fibrosis(UIP/IPF) and 6 controls were used, samples of patients withnon-specific interstitial pneumonia were excluded.

Immune-Related Data Set

Gene expression of 28 immune-related tissues and 118non-immune/non-cancer tissues included in data set GSE1133 (Su et al.,2004) were used in this study.

Validation Data Sets

Data set GSE22459 (Park et al., 2010) contains 65 renal transplantrecipients with signs of fibrosis and inflammation on 1-year protocolbiopsy.

Data set GSE61260 (Hovarth et al., 2014) contains 134 Nash(Non-alcoholic fatty liver disease), PSC (Primary sclerosingcholangitis), PBC (primary biliary cholangitis), NAFLD (Non-alcoholicfatty liver disease), heathy obese and normal liver samples.

Data set GSE48452 (Ahrens et al., 2013) contains 73 samples from Nash(Non-alcoholic fatty liver disease), steatosis, heathy obese and normalliver.

Data set GSE49541 (Moylan et al., 2014) contains 72 patients with NAFLD(40 with mild NAFLD, fibrosis stage 0-1 and 32 with advanced NAFLD,fibrosis stage 3-4).

Data set GSE39491 (Hyland et al., 2014) contains 120 samples ofBarrett's metaplasia and matched normal mucosa from squamous esophagus(NE) and gastric cardia (NC) in 43 BE patients.

Data set GSE26886 (Wang et al., 2013) contains 20 specimens of Barrett'sesophagus patients, 21 specimens of adenocarcinoma patients and 19biopsies from patients with normal esophageal squamous epithelium, 9specimens of squamous cell carcinoma.

Data set GSE37200 (Silvers et al., 2010) contains 31 Barrett's esophagusand 15 adenocarcinoma samples.

Data set GSE47460 (unpublished) contains 582 subjects in total, 254 haveinterstitial lung disease, 220 have COPD, and 108 are controls.

Data set GSE24988 (Mura et al., 2012) contains 116 samples from therecipients organs of PF patients undergoing lung transplantation.

Data set GSE17978 (Emblom-Callahan et al., 2010) contains 58 samplesfrom 12 lungs of patients with end-stage idiopathic pulmonary fibrosisand 6 donors of normal lungs (controls).

Data set GSE53845 (DePianto et al., 2015) contains samples from 40 IPFpatients and 8 healthy controls.

Data set GSE44426 (Desterke et al., 2015) contains 6 bone marrow samplesfrom primary myelofibrosis and 6 control samples.

Two Data Sets of Normal Human Fibroblasts Treated with TGF-β orAbituzumab

The data set comprises expression data for 17 of 19 fibrosis signaturegenes. Expression for three of the 17 genes was below lower limit ofquantification (LLOQ). The first data set (AB001) thus comprisedexpression levels for 14 genes, measured in 9 samples. The nine samplescan be grouped in three sample groups: NHLF alone, NHLF+TGF-β,NHLF+H358. The second data set (AB002) comprises eleven samples: NHLFtreated with 40 ng/ml anti-HEL (3 repeats), NHLF treated with 40 ng/mlAbituzumab (2 repeats), NHLF treated with 10 μg/ml anti-HEL (3 repeats),NHLF treated with 10 μg/ml Abituzumab (3 repeats).

Signature Scoring

We found that our 19-gene and 9-gene signatures can be scored in severalways to yield results that support our claims.

Expression raw intensities can be log.-transformed or not transformed.

Z-normalization: in data sets with multiple samples (>=9) each geneexpression vector can be mean- or median-centered and standardized toyield expression Z scores.

Normalization using pre-treatment intensities as a reference: ratios canbe calculated from expression intensities pre- and post-treatment. Theseratios can be optionally log-scaled.

Summary scores for the signature per sample are calculated as sums,averages, or weighted averages across genes.

Literature-Based Gene List

143 genes associated with up-regulation in SSc and in TGF-stimulatedfibroblasts have been proposed by Daigen Xu (TIP Immunology) includingfour genes from the “Lafyatis signature” that predicts skin disease inpatients with diffuse SSc (Farina et al., 2010). 135 are measured in atleast one of the three SSc data sets. A list of the 135 genes can befound in Table 9.

Computer Programs Used

Program Version Supplier R 3.0.2 R Development Core Team limma R package3.19.24 Bioconductor

Strategy for the Identification of the Desired Fibrosis/SSc Signature

In Figure the strategy for the identification of the desiredfibrosis/SSc signature is outlined. We differ between literature derivedand not-literature derived genes. Both gene sets were tested on thethree SSc data sets, filtered on the pulmonary fibrosis data sets andfinally validated on one more pulmonary fibrosis data set. The singlesteps are described in detail in the next sections.

Step 1: Towards a Pre-Final Gene List

To obtain a candidate list of fibrosis/SSc related genes we performed amoderated t-test for differential expression analysis as implemented inthe R bioconductor package limma (Smyth, 2004) per gene on each SSc dataset. Linear models were fitted to every gene with contrasts “SSc—normal”samples. Given the linear model fit, a moderated t-statistics wascomputed by empirical Bayes moderation of the standard errors towards acommon value. p-values were adjusted for multiple testing by theBenjamini Hochberg procedure (Benjamini and Hochberg, 1995) controllingthe False Discovery Rate (FDR).

Analysis of Literature-Based Genes

All genes were tested for differential expression between SSc and normalskin on the three SSc data sets as described above. Pre-defined genesfrom literature were included in the pre-final list of fibrosis genes ifthey fulfill the following criteria:

-   -   Genes have to be significantly up-regulated (adjusted        p-value<0.05) in at least one of the three public SSc data sets        and    -   genes must not be significantly down-regulated in any of the        three data sets.

None of the genes were excluded due to concurrent significant up- anddown-regulation across the data sets. 40 of the 135 pre-defined genesfrom literature met these criteria.

Additional Fibrosis/SSc Genes Based on Public Data Mining

For all other genes not pre-defined from literature criteria weretightened. Genes were included in the pre-final list of up-regulatedfibrosis/SSc genes if the following criteria are met:

-   -   Genes have to be significantly up-regulated (adjusted        p-value<0.05) in (at least) two of the three public SSc data        sets and    -   genes must not be significantly down-regulated in any of the        three data sets.

62 genes not-derived from literature were found to be up-regulated inSSc compared to healthy skin. Although two of 62 genes were excluded dueto significant down-regulation in one SSc data set, resulting in 60genes that were considered for further analyses.

In total, 100 genes were included in the pre-final list of up-regulatedfibrosis/SSc genes.

Step 2: Filtering of Pre-Final Gene List for Differential Expression inLung Fibrosis

Two data sets, GSE24206 and GSE48149, were used to assess whether thegenes found to be up-regulated in SSc were up-regulated in pulmonaryfibrosis (PF), too. 96 of the 100 genes were present in at least of thetwo PF data sets. We tested the 96 genes in four comparisons forup-regulation in fibrotic lung tissue with moderated t-tests:

-   -   1. GSE24206: normal vs. early IPF    -   2. GSE24206: normal vs. advanced IPF    -   3. GSE48149: normal vs. IPF    -   4. GSE48149: normal vs. SSc-PF

To pass this filtering step a gene needed to be significantlyup-regulated (Benjamini Hochberg adjusted p-value<0.05) in at least twocomparisons and not significantly down-regulated in any comparison. 20candidate genes were left after step 2.

Step 3: Filtering of Pre-Final Gene List for Up-Regulation inImmune-Related Tissues

For the remaining genes from the candidate list of fibrosis/SSc relatedgenes we performed a moderated t-test for differential expressionanalysis as implemented in the R bioconductor package limma per gene onthe normal tissue data set GSE1133. Linear models were fitted to everygene with contrasts “immune—other” samples. Given the linear model fit,a moderated t-statistics was computed by empirical Bayes moderation ofthe standard errors towards a common value. p-values were adjusted formultiple testing by the Benjamini Hochberg procedure controlling theFalse Discovery Rate (FDR). One gene (BIRC3) was excluded due toup-regulation in immune-related vs. other normal (non-cancer) tissuesamples.

The Fibrosis/SSc 19-Gene Signature

By means of prior knowledge from literature and gene expression infopublicly available data sets, we have composed a bona fide fibrosis/SScsignature consisting of 19 non-immune-related genes up-regulated in SScand pulmonary fibrosis. The complete list of genes is shown in Table 9.

Expression data of 19 genes are normalized (per data set), i.e. meancentered and standardized across samples. Mean over 19 genes is used asSignature Score per sample.

The Fibrosis/SSc TGF-β-Up/Abituzumab-Down (TUAD) 9-Gene Signature

We aimed to select genes for the TGF-β-up/Abituzumab-down signature as asubset of genes of the 19 gene signature. Therefore, we conducted thefollowing experiment. Normal human lung fibroblasts (NHLF) weresupplemented with TGF-β and co-cultured with cells from the humanepithelial H358 lung cell line. For 14 of 19 genes of the fibrosissignature we were able to yield expression signals. For a gene to beincluded in the TGF-β-up/Abituzumab-down signature (in the followingtermed TUAD signature) it had to show an up-regulation afteradministration of TGF-β (median fold change across 3 repeatedexperiments). Using this criterion, we selected 9 genes. These 9 genesconstitute our 9-gene TUAD signature (see Table 9 denoted by *).

Further, the final TUAD signature of 9 genes was subjected to anexperiment with normal human lung fibroblasts (NHFL) that wereco-cultured with human H358 cells and treated either with anti-HEL (henegg lysozyme) or Abituzumab at 40 ng/ml and 10 μg/ml (see description ofdata set above).

We found that each of the 9 genes (that were previously found to beup-regulated by TGF-β) could be down-regulated by Abituzumab whencompared to the anti-HEL control. Thus, the TUAD 9-gene signaturerepresents a TGF-β-inducible fibrosis signature that can bedown-modulated by Abituzumab, demonstrated by a) down-regulation of allgenes in the 9-gene signature after Abituzumab treatment compared toanti-HEL, and b) because of the down-regulation of the net TUAD 9-genesignature score after Abituzumab treatment compared to anti-HEL.

Differential expression analysis results of the 19 genes included in theFibrosis/SSc signature are shown in Table 10, Table 11 and Table 12 forthe comparisons of SSc to normal skin, in Table 13 for the comparison ofSSc-PF to normal lung, in Table 14 for the comparison of UIP/IPF tonormal lung, in Table 15 for the comparison of early IPF to normal lung,and in Table 16 for the comparison of advanced IPF to normal lung.

None of the 19 Fibrosis/SSc signature genes is up-regulated inimmune-related vs. non-immune-related tissues (Table 17).

Performance of the 19-Gene Fibrosis/SSc and 9-Gene TUAD Signature

Although, single genes show up-regulated expression like RGS5 in SSccompared to normal skin (FIG. 9) and e.g. COL15A1, COL1A1, COMP, IGFBP2aand SSP1 in IPF and SSc-PF compared to normal lung (FIG. 10-FIG. 14a )in distinct data sets, the signature scores derived from the 19-geneFibrosis/SSc signature and from the 9-gene TUAD signature provide a morerobust signal for monitoring fibrosis in skin (FIG. 14b , FIG. 15a ,FIG. 15b , FIG. 16a and FIG. 16b ), lung (FIG. 17a , FIG. 17b , FIG. 18aand FIG. 18b ), liver (FIG. 19a , FIG. 19b , FIG. 20a , FIG. 20b , FIG.21a and FIG. 21b ) and bone marrow (FIG. 22a and FIG. 22b ).

For esophagus and kidney fibrosis, where no control tissue expressiondata is available, genes included in the 19-gene Fibrosis/SSc and 9-geneTUAD signature are coordinately expressed, denoted by a higher thanrandomly expected Coherence Score (Staub, 2012) obtained by 1000iterations (Table 18).

In liver samples (GSE61260), the signature scores of the 19-geneFibrosis/SSc and of the 9-gene TUAD signature correlate well with theFibrosis score (as described in Hovarth et al., 2014) with Spearmancorrelation coefficients of 0.505 and 0.523, respectively (Table 19).

Conclusions

Because our TUAD multi-gene expression signature can be down-modulatedby treatment with abituzumab, and because it is linked to diseaseseverity and/or degree of fibrosis in various fibrotic diseases (asmentioned in the preceding paragraphs), the TUAD signature can beregarded as an indicator of high clinical need in fibrotic diseases. TheTUAD signature can be used for patient selection for abituzumab therapy:high baseline/pre-treatment levels for the TUAD signature can be used toselect patients for which abituzumab has highest potential to achievetherapy success, since we not only demonstrated that high TUAD signaturestatus is linked to fibrotic disease status, but also that abituzumabcan effectively down-modulate the TUAD signature. In addition, the TUADsignature can be used to monitor fibrosis—as we had demonstrated linksto disease severity, and—because of the demonstrated ability ofabituzumab to down-regulated the TUAD signature—it can therefore also beused to monitor abituzumab effectiveness. Because of the mentionedability of the TUAD signature to serve as a marker predicting therapyresponse, and because of the link to disease severity or presence offibrosis in several fibrotic diseases, we consider abituzumab to be adrug that can be used against all fibrotic diseases in general.

Tables

TABLE 8 Literature-based list of genes up-regulated in SSc and inTGF-stimulated fibroblasts present on at least one of the three SSc datasets Gene name SIGLEC1 sialic acid binding Ig-like lectin 1,sialoadhesin IFI44 interferon-induced protein 44 COMP cartilageoligomeric matrix protein THBS1 thrombospondin 1 SPARC secreted protein,acidic, cysteine-rich (osteonectin) SPP1 secreted phosphoprotein 1 TNCtenascin C FAP fibroblast activation protein, alpha POSTN periostin,osteoblast specific factor ELN elastin SERPINE1 serpin peptidaseinhibitor, clade E (nexin, plasminogen activator inhibitor type 1),member 1 BMP1 bone morphogenetic protein 1 TIMP1 TIMP metallopeptidaseinhibitor 1 TIMP3 TIMP metallopeptidase inhibitor 3 MMP1 matrixmetallopeptidase 1 (interstitial collagenase) MMP2 matrixmetallopeptidase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IVcollagenase) PLAUR plasminogen activator, urokinase receptor PLOD2symbol HAS2 hyaluronan synthase 2 LOX lysyl oxidase LOXL1 lysyloxidase-like 1 TGF-β1 transforming growth factor, beta 1 TGF-β2transforming growth factor, beta 2 TGF-β3 transforming growth factor,beta 3 CYR61 cysteine-rich, angiogenic inducer, 61 CTGF connectivetissue growth factor THBS2 thrombospondin 2 ITGB5 integrin, beta 5 PDGFAplatelet-derived growth factor alpha polypeptide ACTA2 actin, alpha 2,smooth muscle, aorta SNAI1 snail family zinc finger 1 EGR1 early growthresponse 1 EGR2 early growth response 2 CCL2 chemokine (C-C motif)ligand 2 IL6 interleukin 6 (interferon, beta 2) COL9A1 collagen, typeIX, alpha 1 COL1A1 collagen, type I, alpha 1 COL9A3 collagen, type IX,alpha 3 COL6A2 collagen, type VI, alpha 2 COL5A3 collagen, type V, alpha3 COL1A2 collagen, type I, alpha 2 COL27A1 collagen, type XXVII, alpha 1COL13A1 collagen, type XIII, alpha 1 COL4A5 collagen, type IV, alpha 5COL6A6 collagen, type VI, alpha 6 COL18A1 collagen, type XVIII, alpha 1COL14A1 collagen, type XIV, alpha 1 COL4A3BP collagen, type IV, alpha 3(Goodpasture antigen) binding protein COL4A1 collagen, type IV, alpha 1COL10A1 collagen, type X, alpha 1 COL25A1 collagen, type XXV, alpha 1COL3A1 collagen, type III, alpha 1 COL22A1 collagen, type XXII, alpha 1COLEC11 collectin sub-family member 11 COL2A1 collagen, type II, alpha 1COL11A1 collagen, type XI, alpha 1 COL9A2 collagen, type IX, alpha 2COL17A1 collagen, type XVII, alpha 1 COL20A1 collagen, type XX, alpha 1COL4A6 collagen, type IV, alpha 6 COL6A3 collagen, type VI, alpha 3COL4A4 collagen, type IV, alpha 4 COL5A1 collagen, type V, alpha 1 COLQcollagen-like tail subunit (single strand of homotrimer) of asymmetricacetylcholinesterase COL11A2 collagen, type XI, alpha 2 COL6A1 collagen,type VI, alpha 1 COL8A2 collagen, type VIII, alpha 2 COL4A3 collagen,type IV, alpha 3 (Goodpasture antigen) COL4A2 collagen, type IV, alpha 2COL5A2 collagen, type V, alpha 2 COL21A1 collagen, type XXI, alpha 1COLEC12 collectin sub-family member 12 COL29A1 collagen, type XXIX,alpha-1 COL8A1 collagen, type VIII, alpha 1 COLEC10 collectin sub-familymember 10 (C-type lectin) COL19A1 collagen, type XIX, alpha 1 COL15A1collagen, type XV, alpha 1 COL12A1 collagen, type XII, alpha 1 COL16A1collagen, type XVI, alpha 1 COL24A1 collagen, type XXIV, alpha 1 COL23A1collagen, type XXIII, alpha 1 COL7A1 collagen, type VII, alpha 1 MMP9matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa typeIV collagenase) MMP19 matrix metallopeptidase 19 MMP27 matrixmetallopeptidase 27 MMP16 matrix metallopeptidase 16 (membrane-inserted)MMP20 matrix metallopeptidase 20 MMP28 matrix metallopeptidase 28 MMP12matrix metallopeptidase 12 (macrophage elastase) MMP26 matrixmetallopeptidase 26 MMP7 matrix metallopeptidase 7 (matrilysin, uterine)MMP15 matrix metallopeptidase 15 (membrane-inserted) MMP11 matrixmetallopeptidase 11 (stromelysin 3) MMP3 matrix metallopeptidase 3(stromelysin 1, progelatinase) MMP24 matrix metallopeptidase 24(membrane-inserted) MMP21 matrix metallopeptidase 21 MMP14 matrixmetallopeptidase 14 (membrane-inserted) MMP17 matrix metallopeptidase 17(membrane-inserted) MMP8 matrix metallopeptidase 8 (neutrophilcollagenase) MMP13 matrix metallopeptidase 13 (collagenase 3) MMP25matrix metallopeptidase 25 MMP10 matrix metallopeptidase 10 (stromelysin2) MMP23B matrix metallopeptidase 23B ITGA2B integrin, alpha 2b(platelet glycoprotein IIb of IIb/IIIa complex, antigen CD41) ITGAEintegrin, alpha E (antigen CD103, human mucosal lymphocyte antigen 1alpha polypeptide) ITGAM integrin, alpha M (complement component 3receptor 3 subunit) ITGAV integrin, alpha V ITGB1 integrin, beta 1(fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2,MSK12) ITGA3 integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA-3receptor) ITGA1 integrin, alpha 1 ITGB2 integrin, beta 2 (complementcomponent 3 receptor 3 and 4 subunit) ITGB3BP integrin beta 3 bindingprotein (beta3-endonexin) ITGAL integrin, alpha L (antigen CD11A (p180),lymphocyte function-associated antigen 1 alpha polypeptide) ITGB1BP3integrin beta 1 binding protein 3 ITGB3 integrin, beta 3 (plateletglycoprotein IIIa, antigen CD61) ITGB8 integrin, beta 8 ITGA8 integrin,alpha 8 ITGB6 integrin, beta 6 ITGB7 integrin, beta 7 ITGA2 integrin,alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor) ITGB1BP2 integrinbeta 1 binding protein (melusin) 2 ITGA6 integrin, alpha 6 ITGA5integrin, alpha 5 (fibronectin receptor, alpha polypeptide) ITGA10integrin, alpha 10 ITGB4 integrin, beta 4 ITGAX integrin, alpha X(complement component 3 receptor 4 subunit) ITGA9 integrin, alpha 9ITGA7 integrin, alpha 7 ITGAD integrin, alpha D ITGA4 integrin, alpha 4(antigen CD49D, alpha 4 subunit of VLA-4 receptor) ITGB1BP1 integrinbeta 1 binding protein 1 ITGA11 integrin, alpha 11 ITGBL1 integrin,beta-like 1 (with EGF-like repeat domains) CSPG2 chondroitin sulfateproteoglycan 2 ITGB4BP/ eukaryotic translation initiation factor 6 EIF6

TABLE 9 19-gene SSc/fibrosis signature and the 9-gene TUAD signature(genes marked by *) symbol gene name NCBI RefSeq COL15A1 * collagen,type XV, alpha 1 NM_001855 COL1A1 * collagen, type I, alpha 1 NM_000088COMP * cartilage oligomeric matrix protein NM_000095 RGS5 regulator ofG-protein signaling 5 NM_003617 COL10A1 * collagen, type X, alpha 1NM_000493 COL5A1 * collagen, type V, alpha 1 NM_000093 IGFBP2insulin-like growth factor binding NM_000597 protein 2, 36kDa LOXL1lysyl oxidase-like 1 NM_005576 MOXD1 monooxygenase, DBH-like 1 NM_015529ADRA2A adrenoceptor alpha 2A NM_000681 COL5A2 * collagen, type V, alpha2 NM_000393 MMP10 matrix metallopeptidase 10 (stromelysin 2) NM_002425tumor necrosis factor receptor superfamily, NM_014452 member TNFRSF21 21ITGA7 * integrin, alpha 7 NM_002206 TGF-β3 transforming growth factor,beta 3 NM_003239 MMP11 * matrix metallopeptidase 11 (stromelysin 3)NM_005940 SPP1 secreted phosphoprotein 1 NM_000582 CCL2 chemokine (C-Cmotif) ligand 2 NM_002982 TNC * tenascin C NM_002160

TABLE 10 Results of moderated t-tests of 19 fibrosis/SSc signature genescomparing SSc to normal skin samples in GSE45485 Gene symbol logFCp-value adj. p-value COL15A1 −0.05354741 0.63284437 0.85974021 COL1A1−0.0564569 0.32613521 0.65911219 COMP 0.06396207 0.35028311 0.6787201RGS5 0.76552328 8.21E-07 0.00076264 COL10A1 0.14700345 0.155619120.48216536 COL5A1 0.17074828 0.18609626 0.52034919 IGFBP2 0.178494830.07746999 0.3473223 LOXL1 0.00652931 0.95853833 0.98965775 MOXD11.7533069 1.09E-05 0.00250698 ADRA2A 0.58765 0.0010264 0.02967007 COL5A2−0.10143678 0.21608038 0.55557497 MMP10 0.78422241 0.00161561 0.03848804TNFRSF21 0.07153966 0.41999428 0.73134954 ITGA7 0.2628 0.060293340.30433949 TGF-β3 0.16204828 0.07167408 0.33376361 MMP11 0.025765520.87854852 0.9641284 SPP1 0.11211086 0.03788071 0.23926337 CCL20.0468331 0.42387631 0.73379499 TNC 0.0137 0.93305485 0.98151167 logFC =log fold change (from normal skin to SSc)

TABLE 11 Results of moderated t-tests of 19 fibrosis/SSc signature genescomparing SSc to normal skin samples in GSE32413 Gene symbol logFCp-value adj. p-value COL15A1 8.65252005 1.03E-08 7.82E-06 COL1A15.43265134 0.00155515 0.04371919 COMP 14.7144265 5.72E-08 2.83E-05 RGS510.968877 3.13E-10 4.76E-07 COL10A1 15.6671591 0.00031919 0.01472844COL5A1 4.77696658 8.35E-06 0.00103754 IGFBP2 5.12541043 0.000101140.00668004 LOXL1 7.84100134 1.77E-07 6.55E-05 MOXD1 2.292020050.00022047 0.01145535 ADRA2A 8.27779545 0.0013305 0.03929527 COL5A25.45322193 0.00029239 0.01384761 MMP10 3.81981016 0.03358148 0.27003285TNFRSF21 1.99499398 0.00169115 0.04633193 ITGA7 9.61105481 6.16E-060.00087583 TGF-β3 8.35457821 1.64E-05 0.00172723 MMP11 7.378921120.00018876 0.01035532 SPP1 11.7482176 0.00097261 0.03164442 CCL218.0195571 3.18E-12 1.05E-08 TNC 7.44044385 3.28E-05 0.00296034 logFC =log fold change (from normal skin to SSc)

TABLE 12 Results of moderated t-tests of 19 fibrosis/SSc signature genescomparing SSc to normal skin samples in GSE9285 Gene symbol logFCp-value adj. p-value COL15A1 −0.80155204 0.4462029 0.64884688 COL1A12.7221 0.12952504 0.32731037 COMP 13.0156364 0.00032598 0.01168603 RGS50.50973333 0.7285143 0.8421874 COL10A1 0.42446491 0.49108962 0.68406853COL5A1 −0.02584363 0.91217968 0.95182504 IGFBP2 6.09920351 0.001470530.02511234 LOXL1 0.24474737 0.69148258 0.8189216 MOXD1 0.127817540.29020488 0.5176974 ADRA2A NA NA NA COL5A2 1.29160053 0.253038640.47936774 MMP10 −0.02789369 0.54630288 0.72218046 TNFRSF21 0.18278070.00237558 0.03309656 ITGA7 2.09964561 0.12139797 0.31533591 TGF-β30.79875045 0.34075749 0.56391476 MMP11 1.2299009 0.13554094 0.33526052SPP1 5.44541353 0.03795579 0.16699841 CCL2 2.2879614 0.306019350.5326055 TNC 1.03070292 0.03803824 0.16720506 logFC = log fold change(from normal skin to SSc). NA = not available (ADRA2A is not measured inGSE9285)

TABLE 13 Results of moderated t-tests of 19 fibrosis/SSc signature genescomparing SSc-PF to normal lung samples in GSE48149 Gene symbol logFCp-value adj. p-value COL15A1 1.9608 <0.0001 1.00E-04 COL1A1 2.086<0.0001 3.00E-04 COMP 2.5904 <0.0001 1.00E-04 RGS5 1.1167 0.0027 0.017COL10A1 1.3117 1.00E-04 6.00E-04 COL5A1 0.6399 0.0628 0.1754 IGFBP21.5049 <0.0001 1.00E-04 LOXL1 0.3851 0.0102 0.046 MOXD1 0.6583 0.02250.0806 ADRA2A 0.4079 0.022 0.0806 COL5A2 1.1264 0.0032 0.0191 MMP101.3084 0.0042 0.0233 TNFRSF21 1.1712 <0.0001 <0.0001 ITGA7 0.5962 0.00720.0343 TGF-β3 1.3211 <0.0001 4.00E-04 MMP11 1.8952 3.00E-04 0.0026 SPP13.6653 <0.0001 1.00E-04 CCL2 1.6668 0.0019 0.0131 TNC 0.7709 0.00590.0293 logFC = log fold change (from normal lung to SSc-PF)

TABLE 14 Results of moderated t-tests of 19 fibrosis/SSc signature genescomparing UIP/IPF to normal lung samples in GSE48149 Gene symbol logFCp-value adj. p-value COL15A1 1.5947 <0.0001 3.00E-04 COL1A1 2.2934<0.0001 <0.0001 COMP 2.4615 <0.0001 <0.0001 RGS5 1.1082 4.00E-04 0.0027COL10A1 1.1817 <0.0001 <0.0001 COL5A1 0.8224 0.0021 0.0109 IGFBP2 1.2668<0.0001 1.00E-04 LOXL1 0.3737 9.00E-04 0.0056 MOXD1 0.8316 <0.00014.00E-04 ADRA2A 0.1787 0.3115 0.5192 COL5A2 1.0829 1.00E-04 7.00E-04MMP10 1.121 0.0208 0.0761 TNFRSF21 1.0945 <0.0001 <0.0001 ITGA7 0.3660.0074 0.0321 TGF-β3 1.222 <0.0001 1.00E-04 MMP11 2.0049 <0.00011.00E-04 SPP1 3.1784 <0.0001 2.00E-04 CCL2 1.4158 0.0052 0.0236 TNC0.6445 0.0081 0.0333 logFC = log fold change (from normal lung toUIP/IPF)

TABLE 15 Results of moderated t-tests of 19 fibrosis/SSc signature genescomparing early IPF to normal lung samples in GSE24206 Gene symbol logFCp-value adj. p-value COL15A1 2.4835 <0.0001 7.00E-04 COL1A1 1.57051.00E-04 0.0015 COMP 2.0974 4.00E-04 0.0053 RGS5 0.8922 0.002 0.0158COL10A1 1.8361 0.0021 0.0158 COL5A1 0.9854 0.0021 0.0158 IGFBP2 1.43110.0039 0.0218 LOXL1 1.1768 0.0049 0.0258 MOXD1 0.7065 0.0051 0.0258ADRA2A 0.9086 0.0064 0.0308 COL5A2 0.712 0.0092 0.0369 MMP10 1.42970.0725 0.1699 TNFRSF21 0.8836 0.014 0.0539 ITGA7 0.3274 0.1951 0.3345TGF-β3 0.2844 0.0418 0.1143 MMP11 0.0967 0.5179 0.666 SPP1 1.2133 0.09430.2105 CCL2 −1.3519 0.0149 0.0551 TNC 0.2502 0.5779 0.6849 logFC = logfold change (from normal lung to early IPF)

TABLE 16 Results of moderated t-tests of 19 fibrosis/SSc signature genescomparing advanced IPF to normal lung samples in GSE24206 Gene symbollogFC p-value adj. p-value COL15A1 2.5317 <0.0001 2.00E-04 COL1A1 1.70031.00E-04 0.0013 COMP 2.6938 <0.0001 1.00E-04 RGS5 0.9015 6.00E-04 0.0062COL10A1 1.6965 <0.0001 2.00E-04 COL5A1 0.9463 0.0017 0.0136 IGFBP2 2.555<0.0001 <0.0001 LOXL1 1.4254 5.00E-04 0.0055 MOXD1 0.6028 0.0152 0.0729ADRA2A 0.9429 5.00E-04 0.0055 COL5A2 0.6755 0.0108 0.0576 MMP10 2.79841.00E-04 0.0016 TNFRSF21 1.2348 7.00E-04 0.0065 ITGA7 0.7677 0.00630.0435 TGF-β3 0.3516 0.0087 0.052 MMP11 0.117 0.4332 0.6515 SPP1 1.20310.0672 0.1955 CCL2 −0.7953 0.0666 0.1955 TNC 0.2762 0.5107 0.7013 logFC= log fold change (from normal lung to advanced IPF)

TABLE 17 Results of moderated t-tests of 19 fibrosis/SSc signature genescomparing immune-related to non-immune/non-cancer samples in GSE1133Gene symbol logFC p-value adj. p-value COL15A1 −0.57352943 0.004751520.00902789 COL1A1 −0.5599531 0.00583927 0.01008602 COMP −0.422989760.03724964 0.05055309 RGS5 −1.26000495 6.18E-10 5.87E-09 COL10A1−0.38023756 0.06112002 0.06831061 COL5A1 −0.61908543 0.002308960.00548379 IGFBP2 −0.91215384 7.28E-06 3.46E-05 LOXL1 −0.59034430.00366005 0.00772677 MOXD1 −0.70997396 0.00047627 0.00142367 ADRA2A−0.52178229 0.01019951 0.01490697 COL5A2 −0.54032041 0.007810640.01236685 MMP10 −0.08672332 0.66921027 0.70638861 TNFRSF21 −0.030086530.88216925 0.88216925 ITGA7 −0.98300833 1.35E-06 8.54E-06 TGF-β3−1.30537499 1.49E-10 2.82E-09 MMP11 −0.38530818 0.05775116 0.06831061SPP1 −0.8725154 1.78E-05 6.75E-05 CCL2 −0.70471063 0.00052451 0.00142367TNC −0.39344598 0.05266811 0.06671293 logFC = log fold change (fromnon-immune to immune-related samples)

TABLE 19 Spearman correlation between signature scores of the 19-geneSSc/fibrosis signature and the 9-gene TUAD signature and inflammation.NAS (NADLF activity score) and fibrosis score in GSE61260 19-geneSSc/fibrosis 9-gene TUAD signature signature score score Inflammationscore 0.354 0.335 NAS 0.270 0.284 Fibrosis score 0.505 0.523

Bibliography

-   1 Benjamini Y, and Hochberg Y. Controlling the false discovery rate:    a practical and powerful approach to multiple testing. Journal of    the Royal Statistical Society Series B. 1995; 57, 289-300.-   2 Farina G, Lafyatis D, Lemaire R, Lafyatis R. A four-gene biomarker    predicts skin disease in patients with diffuse cutaneous systemic    sclerosis. Arthritis Rheum. 2010 February; 62(2):580-8.-   3 Hinchcliff M, Huang C C, Wood T A, Matthew Mahoney J et al.    Molecular signatures in skin associated with clinical improvement    during mycophenolate treatment in systemic sclerosis. J Invest    Dermatol 2013 August; 133(8):1979-89.-   4 Meltzer E B, Barry W T, D'Amico T A, Davis R D et al. Bayesian    probit regression model for the diagnosis of pulmonary fibrosis:    proof-of-principle. BMC Med Genomics 2011 Oct. 5; 4:70.-   5 Milano A, Pendergrass S A, Sargent J L, George L K et al.    Molecular subsets in the gene expression signatures of scleroderma    skin. PLoS One 2008 Jul. 16; 3(7):e2696.-   6 Pendergrass S A, Lemaire R, Francis I P, Mahoney J M et al.    Intrinsic gene expression subsets of diffuse cutaneous systemic    sclerosis are stable in serial skin biopsies. J Invest Dermatol 2012    May; 132(5):1363-73.-   7 Smyth G K. Linear models and empirical Bayes methods for assessing    differential expression in microarray experiments. Statistical    Applications in Genetics and Molecular Biology. 2004; 3(1), article    3.-   8 Su A I, Wiltshire T, Batalov S, Lapp H et al. A gene atlas of the    mouse and human protein-encoding transcriptomes. Proc Natl Acad Sci    USA 2004 Apr. 20; 101 (16): 6062-7.-   9 Staub E. An interferon response gene expression signature is    activated in a subset of medulloblastomas. Transl Oncol. 2012    August; 5(4):297-304. Epub 2012 Aug. 1

Example 5 Phase I Study Clinical Study

A Phase I trial was initiated to determine safety, pharmacokinetics (andantitumor activity) of CRPC patients treated with DI17E6 includingeffects on prostate-specific antigen, circulating tumor cells (CTC), andsoft tissue and bone metastases. All patients were suffering from aprogressing disease after chemotherapy. Patients were treated withiv-infusions of 250, 500, 1000 or 1500 mg DI17E6 given over 1 hour.

Eligible patients were aged 18 years or older and had histologically orcytologically proven prostate cancer with evidence of bone metastasesafter prior chemotherapy. Patients had either undergone bilateralorchiectomy or were receiving continuous androgen deprivation therapywith a gonadotropin releasing hormone agonist or antagonist and hadstopped anti-androgen therapy for at least 4 weeks prior to enrolment.Patients were required to either be on stable (i.e. at least 3 months)ongoing bisphosphonate therapy or without any bisphosphonate therapy,with a total serum testosterone level less than 50 ng/dL. All patientshad evidence of progressive disease, defined as at least twoprostate-specific antigen (PSA) values above the individual nadir levelwith a minimum increase of 10% each determined at least two weeks priorto screening; nodal or visceral progression was sufficient for inclusionindependent of PSA. In addition, patients had to have an EasternCooperative Oncology Group (ECOG) score of 0 to 2, a life expectancy ofat least 3 months, and adequate hematologic, renal and hepatic function.An institutional review board at each study center approved the studyprotocol, and all patients provided written informed consent.

In this phase 1, multicenter, open-label, dose-escalation study, mCRPCpatients were administered three intravenous infusions of EMD 525797 atdoses of 250, 500, 1000, or 1500 mg given over one hour every two weeksprior to response assessment at the end of week 6. Patients withoutevidence of progressive disease were eligible to receive furtherfortnightly doses until disease progression or unacceptable toxicity.Dose-limiting toxicities (DLTs) were assessed during the first six weeksand patients were followed for safety until four weeks after the lastadministration of EMD 525797. Patients were recruited in four sequentialdose cohorts; after the last of six patients within a dose cohort hadreached the end of week 6, a Safety Monitoring Committee determinedsubsequent dose escalation.

Twenty-six male patients aged between 43 and 80 years (median age, 66years) were enrolled and received at least one intravenous infusion ofEMD 525797, constituting the safety population. All patients were ofCaucasian origin. In general, demographic characteristics werecomparable across the four dose cohorts (Table 1a), with a median timesince first diagnosis of 5.2 years (range, 2-18 years) and a median timefrom diagnosis to first metastatic disease of 0.1 years (range, 0-16years). Two patients withdrew before the end of the DLT period and weresubsequently replaced, with 24 patients receiving three doses of EMD525797 through treatment Week 6.

TABLE 1a Patient baseline demographics (safety population). DI17E6 (EMD525797) dose cohort 250 mg 500 mg 1000 mg 1500 mg Total Characteristic(N = 8) (N = 6) (N = 6) (N = 6) (N = 26) Age, years, mean 67 63 62 66 65(range) (57-78) (47-77) (43-79) (52-80) (43-80) Weight, kg, mean 82.285.8 80.5 93.7 85.3 BMI, kg/m², mean 26.2 28.1 26.0 28.9 27.2 BMI, bodymass index;

In summary (Table 1b ):

Objectives Safety, tolerability and PK after multiple rising i.v. dosesDetermine changes in markers of bone metabolism during treatmentInvestigate changes in efficacy parameters within the whole dose rangeIdentification of potential pharmacodynamic markers Number of Per doselevel n = 6 subjects Dose selection 250, 500, 1000, 1500 mg as 1 h i.v.infusion Treatment 6 weeks (every second week) and 4 weeks follow up.duration Option for further treatment if patient is non-progressive.Dose escalation Dose escalation will be based on the toxicity assessment(DLT) after 6 weeks.

The clinical results from this Phase I clinical trial is being conductedat 3 sites in Germany and at one site in Belgium.

Treatment Duration

24 patients (43-80 years) received 3 doses (weeks 1, 3 and 5) prior toresponse assessment at the end of week 6. Dose-limiting toxicities(DLTs) were assessed over the first 6 weeks and patients were followedfor safety until 4 weeks after the last administration of DI17E6.

Table 2a summarizes drug exposure per patient in each cohort. Patientshad a mean EMD 525797 exposure duration of 117.5 days (median, 74.5days; range, 14-534 days). Thirteen of 24 patients had a longer exposuretime than expected (>84 days), with two patients in the 500 mg cohortremaining on treatment for 297 and 534 days, and one patient in the 1000mg cohort receiving treatment for 310 days. No DLTs were reported withinthe DLT period of 6 weeks. All patients experienced at least one is TEAEand no dose-dependent relationship in TEAEs was observed.

TABLE 2a DI17E6 exposure per patient in each of the dose cohorts Pt 250mg 500 mg 1000 mg 1500 mg 1 42 297 113 91   2 42   380+ 121 84+ 3 42 85  198+ 72+ 4 42 142 41 64+ 5 56 140 56 77+ 6 98 56 43 57+ 7  14* 8 28* + = ongoing treatment; *dropped out pts (1 and 2 infusions only)

The study protocol stated, that subjects will receive every other weekat least 3 doses (250, 500, 1000 mg/each 2 weeks) of DI17E6.

TABLE 2b This table shows the values of the treatment duration (state:August 2010). Dose Patients/No. weeks treatment Level 1001 1002 10031004 1005 1006 1008 1010 2001 2002 2003 2005 2007 2009 1 8.3 8.3 3.3 8.06.1 9.1 4.1 13.0 2 42.3 49.3 12.1 18.3 20.0 5.4 3 4 Dose Patients/No.weeks treatment Level 3001 3002 3003 3004 3005 3006 4002 4003 4004 40054009 4011 1 2 3 14.1 17.5 24.3 8.0 7.1 8.1 4 9.4 6.4 8.3 9.1 8.1 2.43Dose level 1: 250 mg Dl17E6; dose level 2: 500 mg Dl17E6; dose level 3:1000 mg Dl17E6, and dose level 4: 1500 mg Dl17E6.

Safety/Side Effects

Table 3 summarizes drug-related TEAEs. Eleven patients (42.3%)experienced drug-related TEAEs, which were most commonly reported in thesystem organ class “Skin and subcutaneous tissue disorders” (4 patientstotal; pruritus generalized, erythema, rash), “General disorders andadministration site conditions” (3 patients total; fatigue, mucosalinflammation, edema peripheral), “Gastrointestinal disorders” (2patients total; dry mouth, swollen tongue, upper gastrointestinalhemorrhage), and “Infections and infestations” (2 patients total;rhinitis, septicemia). Only two patients (7.7%) had a drug-related grade3 or 4 TEAE: one patient in the 500 mg cohort experienced a grade 3increase of gamma-glutamyltransferase (GGT) and a single patient in the1000 mg cohort experienced a grade 3 septicemia.

Two patients experienced serious TEAEs that were considered to berelated to treatment. These were a grade 1 upper gastrointestinalhemorrhage in a patient in the 250 mg cohort and a grade 3 septicemia inone patient in the 1000 mg cohort. At screening, this latter patient hadan ongoing diagnosis of urinary tract infection and recurrent events ofsepticemia of which the last event was considered related to DI17E6 (EMD525797). Four patients died and investigators assessed all deaths as notreasonably related to EMD 525797.

Six patients (23.1%) permanently discontinued treatment due to TEAEs,including 4 patients in the 250 mg cohort (upper gastrointestinalhemorrhage at day 12, muscular weakness at day 53, paraplegia at day 46,and ureteric obstruction at day 30), and 1 patient each in the 500 mg(grade 3 GGT increase at day 534) and 1500 mg (metastases to centralnervous system at day 85) cohorts. No administration site skin reactionswere reported. Post-baseline hematology and biochemistry toxicity shiftsto grade 3 or 4 occurred in 8 patients. However, there were no obvioustrends in laboratory findings, vital signs or ECG recordings. Overall,65.4% of patients had the same worst ECOG performance status ontreatment compared with baseline.

TABLE 3 Drug-related treatment emergent adverse events* (TEAEs). TEAEsrelated to EMD 525797 n (%) Patients with events 11 (42) Skin andsubcutaneous tissue disorders  4 (15) Pruritus generalized 2 (8)Erythema 1 (4) Rash 1 (4) General disorders and administration site  3(12) conditions Fatigue 1 (4) Mucosal inflammation 1 (4) Edemaperipheral 1 (4) Gastrointestinal disorders 2 (8) Dry mouth 1 (4)Swollen tongue 1 (4) Upper gastrointestinal hemorrhage 1 (4) Infectionsand infestations 2 (8) Rhinitis 1 (4) Septicemia^(t) 1 (4) Eye disorders1 (4) Vision blurred 1 (4) Investigations 1 (4) Blood pressure increase1 (4) Musculoskeletal and connective tissue disorders 1 (4) Arthralgia 1(4) Nervous system disorders 1 (4) Dysgeusia 1 (4) Respiratory,thoracic, and mediastinal disorders 1 (4) Dyspnea 1 (4) *MedDRA PrimarySystem Organ Class. MedDRA Preffered Term Version 13.0. ^(t)Grade 3occurred after the first 6 weeks (dose limiting toxicity period).

Examples of Observed Side Effect:

(i) A 62 year-old male experienced grade 1 upper gastrointestinalbleeding 13 days after the first and only infusion of DI17E6 (250 mg).The subject presented with non-serious hematemesis and was hospitalized.Gastroscopy showed a lesion in the distal esophagus. Active bleeding wasexcluded, the subject was treated with omeprazole, and the eventresolved(ii) A 79 year-old patient developed septicemia (grade 2) due to e.faecalis 1 days after the most recent and 9 weeks after the firstinfusion of DI17E6 (EMD 525797) (1000 mg). The patient was hospitalizedand discharged with recovery. A month later, the patient developed asecond septicemia episode (grade3) again due to e. faecalis 4 days afterthe most recent and 2.5 months after the first infusion. The patient washospitalized and discharged with recovery. Another month later, thepatient developed a third septicemia episode (grade 3) 4 days after themost recent and 3.5 months after the first infusion, which wasattributed to EMD 525797.

In Summary:

-   -   Accumulating safety data have been reviewed at all 4 SMCs    -   Overall no DLTs have been observed: DLT is defined as any grade        3 or 4 hematological or non-hematological toxicity occurring        until end of week 6 suspected to be reasonably related to the        investigational product by the Investigator and/or Sponsor        except for allergic/hypersensitivity reactions and any        out-of-range laboratory values without clinical correlate which        are reversible within 7 days.    -   No MTD reached until now    -   Overall only 2 SAEs have been observed as related to study        medication

Pharmacokinetics and Pharmacodynamics

After single and multiple doses, EMD 525797 showed a dose-dependent,non-linear PK profile. After the first 1-hour intravenous infusion,C_(max) of DI17E6 (EMD 525797) was generally reached within 1-2 hoursafter the start of dosing. The elimination half-life increased with doseas a consequence of EMD 525797 clearance increasing with dose, whereasmean volume of distribution remained constant over the dose range (Table4).

As given in Table 4 below, administration of cohort 2 CRPC patients with500 mg/each 2 weeks reached serum levels with IC95, whereas patientsfrom cohort 1 with 250 mg/each 2 weeks failed. The serum troughconcentration of EMD 525797 in cohort 2, 500 mg/each 2 weeks, is abovethe IC₉₅ and reach the IC₉₉ of the non-linear CL pathway (250 mg/each 2weeks failed).

TABLE 4 250 mg 500 mg 1000 mg 1500 mg (N = 6) (N = 6) (N = 6) (N = 6)C_(max′) μg/mL, mean ± SD 57.1 ± 13.8 131.9 ± 22.8  376.6 ± 64.1  498.8± 132.8 T_(max′) h, median (range) 1 (1-5) 3 (1-5) 1 (1-4) 1 (1-8)C_(min′) μg/mL, mean ± SD 2.7 ± 4.1 28.0 ± 12.3 102.8 ± 28.2  150.7 ±47.4  Vss_(′) L, mean ± SD 4.75 ± 1.19 4.35 ± 0.17 3.36 ± 0.36 4.46 ±1.22 AUC_(T′) μg/mL * h, mean ± SD 6694 ± 3746 21225 ± 6505  68145 ±12811 87535 ± 21575 Ratio_AUC, mean ± SD 1.42 ± 0.46 1.37 ± 0.39 1.70 ±0.36 1.70 ± 0.18 Ratio_C_(max′) mean ± SD 1.11 ± 0.17 1.21 ± 0.22 1.25 ±0.12 1.33 ± 0.18 AUC_(T′), area under the concentration-time curvewithin one complete dosing interval; C_(max′) maximum serumconcentration. C_(min′) through serum concentration; Ratio_AUC, relativearea under the curve [AUC_(T)(Dose period3)/AUC_(T)(Dose period 1)];Ratio_C_(max′) relative maxiumum serum concentration [C_(max)(Doseperiod 3)/C_(max)(Dose period 1)]; t_(max)' time to reach C_(max′);V_(ss′) apparent volume of distribution at steady stage.

After multiple doses, DI17E6 (EMD 525797) maximal serum concentrationsand exposure accumulated dose-dependently up to a maximum value (dosingperiod 3/dosing period 1) of 1.33 and 1.70, respectively, at 1500 mg.

In most patients, CTC concentrations remained stable around baselinevalues (data not shown). In two patients, considerable decreases in CTCconcentrations were observed at around 14 and 42 days after the start oftreatment, respectively.

Anti-EMD 525797 antibodies were detected in 4 of 25 (16.0%) evaluablepatients. All four patients were in the 250 mg cohort; two patientsreverted to seronegative status after two weeks, one patient had nofollow-up, and one patient remained seropositive over the entire studyperiod.

1: The anti-αv integrin antibody DI17E6, or a biologically activevariant or modification thereof, for use in the treatment of patientssuffering from fibrosis and/or fibrotic disorders. 2: The anti-αvintegrin antibody DI17E6, or a biologically active variant ormodification thereof, for use according to claim 1, wherein the organsaffected by said fibrosis and/or fibrotic disorders are selected fromthe group consisting of lung, liver, kidney, cardiovascular system orskin. 3: The anti-αv integrin antibody DI17E6, or a biologically activevariant or modification thereof, for use according to claim 1, whereinsaid fibrotic disorder affects one or more organs selected from thegroup consisting of lung, liver, kidney, heart and skin. 4: The anti-αvintegrin antibody DI17E6, or a biologically active variant ormodification thereof, for use according to claim 1, wherein saidfibrotic disorder comprises or is systemic sclerosis (SSc). 5: Theanti-αv integrin antibody DI17E6, or a biologically active variant ormodification thereof, for use in the treatment of patients sufferingfrom systemic sclerosis (SSc). 6: The anti-αv integrin antibody DI17E6,or a biologically active variant or modification thereof, for useaccording to claim 4, wherein the systemic sclerosis comprises systemicsclerosis of the lung, liver, kidney, cardiovascular system or skin. 7:The anti-αv integrin antibody DI17E6, or a biologically active variantor modification thereof, for use according to of claim 4, wherein thesystemic sclerosis affects one or more organs selected from the groupconsisting of lung, liver, kidney, heart and skin. 8: The anti-αvintegrin antibody DI17E6, or a biologically active variant ormodification thereof, for use according to claim 4, wherein the systemicsclerosis affects the cardiovascular system, the blood vessels and/orthe blood. 9: The anti-αv integrin antibody DI17E6, or a biologicallyactive variant or modification thereof, for use according to claim 1,wherein the fibrotic disorder and/or the systemic sclerosis comprisesone or more indications selected from the group consisting of idiopathicpulmonary fibrosis, primary sclerosing cholangitis, non-alcoholicsteatohepatitis (NASH), primary focal glomerulosclerosis, primarysegmental glomerulosclerosis, diabetic nephropathy, diastolicdysfunction and myelofibrosis. 10: The anti-αv integrin antibody DI17E6,or a biologically active variant or modification thereof, for useaccording to claim 1, wherein said fibrotic disorder and/or systemicsclerosis comprises pulmonary fibrosis and/or alveolitis (interstitiallung disease, ILD). 11: The anti-αv integrin antibody DI17E6, or abiologically active variant or modification thereof, for use accordingto claim 1, wherein the disease to be treated is systemic sclerosis ofthe lung and/or skin. 12: The anti-αv integrin antibody DI17E6, or abiologically active variant or modification thereof, for use accordingto claim 11, wherein the systemic sclerosis of the skin is diffusecutaneous systemic sclerosis (dcSSc) or limited cutaneous systemicsclerosis (lcSSc). 13: The anti-αv integrin antibody DI17E6, or abiologically active variant or modification thereof, for use in thetreatment of pulmonary fibrosis, alveolitis (interstitial lung disease,ILD), and/or sclerodermal interstitial lung disease (SSc-ILD). 14: Theanti-αv integrin antibody DI17E6, or a biologically active variant ormodification thereof, for use according to claim 1, wherein saidtreatment comprises the administration of a dose, preferably aneffective dose, of said antibody, or biologically active variant ormodification thereof, in an amount of 500 mg-3000 mg per month. 15: Theanti-αv integrin antibody DI17E6, or a biologically active variant ormodification thereof, for use according to claim 1, wherein saidtreatment comprises the administration of a dose, preferably aneffective dose, of said antibody, or biologically active variant ormodification thereof, in an amount of 1000 mg-2000 mg per month. 16: Theanti-αv integrin antibody DI17E6, or a biologically active variant ormodification thereof, for use according to claim 14, wherein the dose,preferably the effective dose, is administered in a single dose. 17: Theanti-αv integrin antibody DI17E6, or a biologically active variant ormodification thereof, for use according to claim 1, wherein saidtreatment comprises the administration of said antibody, or biologicallyactive variant or modification thereof, in an amount of about 500 mg permonth, about 1000 mg per month, about 1500 mg per month, about 2000 mgper month or about 2500 mg per month. 18: The anti-αv integrin antibodyDI17E6, or a biologically active variant or modification thereof, foruse according to claim 17, wherein said amount is administered in asingle dose. 19: The anti-αv integrin antibody DI17E6, or a biologicallyactive variant or modification thereof, for use according to claim 1,wherein said antibody or said biologically active variant ormodification thereof is administered as monotherapy. 20: The anti-αvintegrin antibody DI17E6, or a biologically active variant ormodification thereof, for use according to claim 1, wherein saidbiological active variant or modification comprises the CDR regions andheavy and light chain variable regions of DI17E6, which are at least 80%identical, at least 90% identical, at least 95% identical, at least 98%identical, or at least 99% identical in amino acid sequence compared tothe variable regions of DI17E6. 21-46. (canceled)